Kits for detection of ATP

ABSTRACT

Methods and kits for detecting the presence of ATP, for measuring ATP concentrations, and for detecting viable cells using a composition comprising an ATP-dependent enzyme and one or more ATPase inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/813,279 entitled METHOD FOR MEASUREMENT OF ATP, filed on Mar. 19,2001, which claims the benefit of U.S. Provisional Patent Application60/269,526 filed on Feb. 16, 2001. Both of these priority patentapplications list Keith Wood, Rita Hannah, and Richard A. Moravec asinventors.

FIELD OF THE INVENTION

The present invention relates generally to the fields of cell biologyand molecular biology. In particular, this invention relates to methods,compositions and kits for improving the detection and quantitation ofATP.

BACKGROUND OF THE INVENTION

Advances in the biological, biomedical and pharmaceutical sciences haveaccelerated the pace of research and diagnostics unparalleled in thepast. With whole genome sequences becoming quickly and successivelyavailable, the assembly of large libraries of small molecules, and theability to move pharmaceutical development, clinical diagnostic testsand basic research from a reductionist to a whole system approachdemands assays that facilitate high throughput analyses. Molecules nolonger need to be singly analyzed for their effects on a lone process;instead, the effects of many molecules on several biological systems canbe studied simultaneously—if appropriate, fast, reliable, and accurateassays are available.

Preferred bioassays that assist in evaluating cellular health are thosethat detect and quantify adenosine triphosphate (ATP). Hydrolysis of ATPpowers many of a cell's biochemical processes. Healthy, viable cells arerich in ATP; dead or dying cells are ATP-poor.

Efficient, reliable and accurate assays for cell viability can be usedto rapidly discover cytotoxic agents or cell proliferation agents anddetermine the cytotoxic effect or cell proliferation effect of agents oncells. Cancer pharmaceutical research often endeavors to identifycompounds that selectively kill quickly-dividing cells—a primarycharacteristic of cancer cells. While some effective anti-cancercytotoxic compounds have been identified; innumerable potentially morevaluable compounds await identification. High throughput screens ofcompound libraries, coupled with efficient cell viability assays, canswiftly identify such compounds. In some systems of the body, controlledcell death is crucial for appropriate function. For example, immunesystem development—a continual process—depends on apoptosis (programmedcell death). The discovery of drugs to treat immuno-related dysfunctionsoften depends on determining cell viability. The efficacy of a candidatecompound on cell viability can be assayed by detecting ATP, since ATPproduction is only realized in metabolically active (live) cells andresidual ATP in a cell is degraded upon cell death, particularly quicklyin non-apoptotic (necrotic) cell death. Assay systems that not onlyfacilitate the evaluation of a substance on cell viability, but alsopermit high throughput screens that can rapidly test thousands ofcompounds, streamline new drug discovery.

In clinical settings, diagnostic tests on large numbers of samples arefacilitated when simple, accurate and safe assays are used. Diseasetreatments can then be more readily determined and instituted.

With the availability of whole genome sequences, the identification ofgene products that affect ATP production, either indirectly or directly,is made possible, and high throughput screens to identify such proteinsare facilitated by simple, fast, accurate and reliable ATP assays.

ATP assays are valuable for innumerable types of measurements for whichit is important to determine the presence or absence of microbes or todetermine the amount of microbial contamination present, e.g.,determining microbial contamination of end products, hygiene monitoring,effectiveness of biocides, success of biological waste treatmentprocesses, and the like.

ATP assays depend on reporter molecules or labels to qualitatively orquantitatively monitor ATP levels. Reporter molecules or labels in suchassay systems have included radioactive isotopes, fluorescent agents,and enzymes, including light-generating enzymes such as luciferase.Desirable characteristics of any reporter molecule systems include safe,quick and reliable application and detection. Luminescent systems areamong the most desirable since they are exceptionally safe andsensitive.

Light-generating enzymes have been isolated from certain bacteria,protozoa, coelenterates, mollusks, fish, millipedes, flies, fungi,worms, and crustaceans. Those enzymes isolated from beetles,particularly the fireflies of the genera Photinus, Photuris, andLuciola, and from click beetles of genus Pyrophorus, have foundwidespread use in reporter systems. In many of these organisms, enzymessuch as luciferases catalyze oxido-reductions in which the free energychange excites a substrate molecule to a high-energy state. When theexcited molecule returns to the ground state, visible light is emitted,i.e. “bioluminescence” or “luminescence.” Among the assay systems inwhich bioluminescence has been employed to monitor or measure ATP arethose in which the activity of an ATP-dependent bioluminescent enzyme,e.g. a beetle luciferase, is exploited.

When luciferase is combined with a sample for the purpose of detectingATP, it is typically desirable to inhibit ATPases endogenous to thesample as well as enzymes that generate ATP, thus assuring that the ATPdetected corresponds to the actual amount of ATP in a sample at adesired time. Many ATPase inhibitors are known, including detergents,especially detergents that are positively charged. However, most ATPaseinhibitors are effective in not only eliminating ATPase functionendogenous to the sample (e.g., a cell or cell population), but alsoATPases that may be used as the reporter molecule, such as luciferase.Additionally, to counter ATP production, inhibitors of enzymes thatphosphorylate, such as kinases, are desirable. However, theseinhibitors, such as sodium fluoride (NaF), might also affect luciferasefunction. A challenge to improving ATP detection in a sample usingluciferase depends on methods or compositions that substantiallydecrease or eliminate ATPase activity and ATP-generating activityendogenous to the sample, thereby stabilizing the amount of ATP presentin the sample to that present when the composition is added, withoutconfounding luciferase function.

There are multiple variations of cellular ATP detection methodscurrently used, all of which act in a stepwise manner. Some such methodsfirst lyse the cells and inactivate the ATPase activity endogenous tothe sample (e.g., by increasing sample pH), then neutralize the ATPaseinhibitor, thereby converting the environment of the sample to onefavorable to luciferase activity prior to adding the luciferase anddetecting luminescence. Other such methods combine the neutralization ofthe ATPase inhibitor with the addition of luciferase. There are no ATPdetection systems that provide a composition or method capable ofinactivating endogenous ATPase activity and detecting luciferaseactivity in the same environmental milieu. Therefore, current assaysthat use luminescence to detect ATP are handicapped by the need forsuccessive, time-consuming steps.

The present invention provides compositions with properties of enhancedstability comprising a luciferase and one or more ATPase inhibitors andfurther provides methods using these novel compositions to detect ATP ina sample by reducing the steps of cell lysis, endogenous ATPaseinhibition, and substrate and luciferase addition to a single step thatis then followed by detection of luminescence. Because embodiments ofthe invention significantly reduce the time and effort ofluciferase-mediated detections of ATP by eliminating the need toneutralize ATPase inhibitor activity before adding luciferase, highthroughput assays can finally be efficiently realized.

SUMMARY OF THE INVENTION

The invention is drawn to methods, compositions and kits that are usedto detect and quantify ATP levels in a sample. The method comprisesadding to a sample a composition (“reagent composition”) comprising aluciferase enzyme and an ATPase inhibitor, and detecting luminescenceproduced in the sample by the conversion of a substrate into aluminescing compound by luciferase. The reagent composition hasproperties of enhanced stability, thereby eliminating the traditionalstep of inhibiting ATPases endogenous to a sample before addingluciferase enzyme to the sample. Thus, although luciferase functions asan ATPase, while in the reagent composition it is resistant to theeffects of an ATPase inhibitor also present in the reagent composition.Such stable reagent compositions facilitate many ATP detections in asample over a long period of time as well as detection of ATP in manysamples over a long period of time.

In general, the methods comprise adding a composition (“reagentcomposition”) comprising a luciferase (such as exemplified by, but notlimited to, SEQ ID NOs.: 1-4) and one or more ATPase inhibitors to asample and detecting luminescence, where the activity of the reagentcomposition has enhanced stability [i.e., the reagent composition iscapable of maintaining at least about 30%, more preferably at leastabout 60% activity (as measured by luminescence when the reagentcomposition is combined with the sample) for at least one hour, evenmore preferably at least 70%, 80%, 90%, 95%, 99% or greater activity forat least one hour, still more preferably for at least two hours and evenmore preferably for at least four hours relative to the reagentcomposition's activity when it is created, i.e., just after (0 to 10minutes) the luciferase enzyme is combined with an ATPase inhibitor],and where the ATPase inhibitor is present in the reagent composition ata concentration sufficient to reduce ATPase activity endogenous to thesample by at least about 25%, more preferably at least about 30%, morepreferably at least about 40%, even more preferably 50%, 60%, 70%, 80%,90%, 95%, or 99% or greater relative to the sample's ATPase activity inthe absence of the ATPase inhibitor. The reagent composition may beadmixed before use by adding a solution comprising one or more ATPaseinhibitors to a lyophilized luciferase.

Loss of stability is defined as irreversible loss of activity. Thereagent composition loses stability over time and the amount of activitylost varies depending on the particular luciferase, ATPase inhibitorand, when present, enzyme stabilizing agent used. Preferably thestability of the reagent composition is demonstrable in the temperaturerange of about 20° C. to about 37° C. Although the methods of theinvention may be used with a sample containing any amount of ATP, it ispreferable to use a sample containing a non-saturated amount of ATP(i.e., a range where luminescence is linearly proportional to theconcentration of ATP).

The luminescence generated by a luciferase reaction is typicallydetected with a luminometer although other detection means may be used.The presence of light greater than background level indicates thepresence of ATP in the sample. The background level of luminescence istypically measured in the same matrix in which the sample exists, but inthe absence of the sample. Suitable control reactions are readilydesigned by one of skill in the art. Preferred luciferases used in thecompositions and methods of the invention generate a stable signal,i.e., they yield enhanced duration of luminescence in a luciferasereaction defined as less than 50% loss of luminescence per hour relativeto the luminescence at the time the luciferase reaction was initiated.Preferred luciferases of the invention allow for multiple analyses of asample over time or analysis of many samples over time, one hour afterthe luciferase is combined with the ATPase inhibitor, more preferablytwo hours and most preferably four hours or more. Optionally, theluciferases used in the compositions and methods of the invention haveenhanced thermostability properties.

Quantifying the amount of emitted light also quantifies the amount ofATP in a sample, and thereby the quantity of living cells. QuantitativeATP values are realized, for example, when the quantity of light emittedfrom a test sample is compared to the quantity of light emitted from acontrol sample or to a standard curve determined by using known amountsof ATP and the same luciferase, substrate, and reaction conditions (i.e.temperature, pH, etc.). It is understood that quantification involvessubtraction of background values. Qualitative ATP values are realizedwhen the luminescence emitted from one sample is compared to theluminescence emitted from another sample without a need to know theabsolute amount of ATP present in the samples, e.g., a comparison ofsamples in the presence or absence of a test compound. Many suchexperiments can readily be designed by one of ordinary skill in the art.

Examples of ATPase inhibitors include detergents, preferably detergentswith charged groups such as cationic detergents [e.g., DTAB(dodecyltrimethylammonium bromide), CTAB (cetyltrimethylammonium) andBDDABr (benzyldimethyldodecylammonium bromide)], anionic detergents(e.g., SDS and deoxycholate), and zwitterionic detergents (e.g.,sulfobetaine 3-10). To facilitate the method, a substrate for theluciferase, such as luciferin, may be included in the reagentcomposition. Other embodiments of the reagent composition furthercomprise a compound, such as NaF, that prevents an increase in ATPlevels in the sample over time. Other compounds that prevent an increasein ATP levels in the sample include vanadate andparanitrophenylphosphate. Still other embodiments of the reagentcomposition further comprise a buffer and magnesium. One of skill in theart knows that other cations, such as manganese and calcium, may besuitable substitutes for magnesium.

The reaction composition may also comprise an enzyme stabilizing agent.The enzyme stabilizing agent can be any compound that stablizes theluciferase from degradation. Suitable enzyme stabilizing agents includeproteins (such as bovine serum albumin or gelatin) or detergents(preferably non-ionic detergents, most preferably THESIT®).

Because the presence of ATP (or a particular ATP:ADP ratio) is aproperty of living cells, the invention is also directed to detectingand quantifying live cells in a sample using the above-describedcompositions. The amount of luminescence then correlates to the numberof viable cells within a population, usually measured by lysing analiquot of a population of cells of interest while applying theinvention or extracting the ATP from a cell or population of cells.

Further, the present invention is useful for determining the effect ofsmall molecules (including organic and inorganic molecules and syntheticand naturally occurring molecules) on living cells, which in turn allowsthe assessment of whether the small molecule may function as apharmaceutical. Thus, the invention is also directed to methods thatdetermine the effect of a compound on a first population of cells bycontacting the first population of cells with a concentration of thecompound and then at a later time contacting the first population ofcells with a composition of the invention, detecting and comparing theamount of luminescence in the first population to an amount ofluminescence in a second population of cells. The second population ofcells may be contacted with a concentration of the compound that is lessthan the concentration contacting the first population of cells or withno compound. A lesser amount of luminescence detected from the firstpopulation compared to the second population may indicate that thecompound comprises a cytotoxic agent. In this way, cytotoxic reagentsmay be discovered. Similarly, the invention is useful for discoveringcell proliferation reagents, i.e., compounds that stimulate cell growth.Using the above example, a lesser amount of luminescence detected fromthe second population compared to the first population may indicate thatthe compound comprises a cell proliferation agent. The invention isuseful for comparing the effects of different compounds at the sameconcentration on cells. The invention is also useful for comparing theeffect of a compound on different types of cells. One of skill in theart may develop many other such assays for which the invention isuseful.

The invention also assembles the elements of the invention into kits.Such kits are designed to determine the presence of ATP in a sample,e.g. measuring cell viability within a population of cells, ordetermining the effects of compounds on cells. Kits can bemultifunctional such that more than one purpose can be realized. In oneembodiment, a kit that is used to detect ATP in a sample may compriselyophilized luciferase in one container, while another containercontains reconstitution buffer with one or more ATPase inhibitors. TheATPase inhibitors may be detergents, preferably detergents with ionicgroups including cationic detergents (preferably DTAB or BDDABr),anionic detergents (preferably SDS or deoxycholate) or zwitterionicdetergents (preferably sulfobetaine 3-10) or a combination thereof.

The kit may also supply a luciferase substrate, such as luciferin. Thekit may also supply magnesium or other cations such as manganese orcalcium. To facilitate the use of control experiments with knownconcentrations of ATP, such as in embodiments of the kits that are usedto quantify ATP in a sample, a container that has ATP may also besupplied in such kits. The kit may also supply a compound that preventsan increase in the amount of ATP in the sample over time (e.g., NaF).The kit may also supply a cell-lysing agent or an ATP extracting agent(e.g., TCA, DMSA, CTAB, ethanol, and the like). The kit may also supplya buffer. The kit may also supply an enzyme stabilizing agent, e.g., BSAor gelatin or THESIT®.

A preferred embodiment of the kit contains components which, whencombined, generate a reagent composition that (i) maintains at leastabout 30% (preferably at least about 60%, even more preferably at least70%, 80%, 90%, 95%, 99%) activity for at least about one hour(preferably at least two hours, more preferably four hours), as detectedby luminescence when the reagent composition is combined with a sample,and relative to the reagent composition's activity just after it isassembled (i.e., 0 to 10 minutes after the component comprisingluciferase is combined with the component comprising an ATPaseinhibitor) and (ii) reduces at least about 25% or at least about 30%,(preferably at least about 40%, even more preferably at least about 50%,60%, 70%, 80%, 90%, 95%, 99% or any increment therein) of the ATPaseactivity that is endogenous to the sample relative to the sample'sATPase activity in the absence of the ATPase inhibitor.

The component comprising an ATPase inhibitor may comprise greater thanone ATPase inhibitor where they are present in the reagent compositionat a concentration such that their combined effect reduces at leastabout 25% or at least about 30%, (preferably at least about 40%, evenmore preferably at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or anyincrement therein) of the ATPase activity that is endogenous to thesample relative to the sample's ATPase activity in the absence of theATPase inhibitor and when allow for the reagent composition.

Most preferably the kit comprises a container comprising a buffereddetergent solution, said buffered detergent solution at a pH in therange of about pH 6.0 to about pH 8.0, and said buffered detergentsolution comprising DTAB whose concentration in the reagent compositionis in the range of about 0.05% to about 2% (w/v) and optionallycomprising NaF whose concentration in the reagent composition is in therange of about 1 mM to about 20 mM and optionally comprising THESIT®whose concentration in the reagent composition is in the range of about1% to about 5%. The kit additionally comprises a separate containercomprising lyophilized luciferase, preferably a luciferase with thesequence of SEQ ID NOs.: 1, 2, 3, or 4, most preferably SEQ ID NOs.: 2or 4. Preferably the luciferase, when combined with the buffereddetergent solution creating the reagent composition, is at aconcentration of 1 μg/ml or greater, more preferably at a concentrationof 80 μg/ml or greater. Preferably, the container comprising lyophilizedluciferase further comprises lyophilized luciferin. Optionally, the kitfurther comprises instructions for use of the kit for the purpose ofmeasuring ATP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the effect of increasing concentrationsof various nonionic and zwitterionic detergents on relative ATPaseactivity in a sample.

FIG. 2 is a graph illustrating the effect of increasing concentrationsof various cationic or anionic detergents on relative ATPase activity ina sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions with properties of enhancedstability comprising a luciferase and one or more ATPase inhibitors. Theinvention further provides compositions with properties of enhancedstability comprising a cell lysing agent, a luciferase and one or moreATPase inhibitors. The invention further provides methods using thesenovel compositions to detect ATP in a sample by reducing the steps ofcell lysis, endogenous ATPase inhibition, and substrate and luciferaseaddition to a single step that is then followed by detection of theresulting luminescence. Alternatively, cell lysis may be replaced withextraction of ATP from a cell or a population of cells. Preferably theluminescence resulting from the combination of a composition of theinvention with a sample has an extended duration, i.e., diminished byless than about 50% per hour relative to the luminescence just after thecomposition is combined with the sample. The process of the inventionsignificantly reduces the time and effort of luciferase-mediateddetection of ATP in a sample by eliminating the need to neutralizeATPase inhibitor activity before adding luciferase.

There are multiple variations of ATP detection methods currently used,all of which act in a stepwise manner. Some such methods first lyse thecells and inactivate the ATPase activity endogenous to a sample (e.g.,by increasing sample pH), and then neutralize the ATPase inhibitor,thereby converting the environment of the sample from one favoringATPase inhibition and unfavorable to luciferase activity to onefavorable to luciferase activity prior to adding luciferase andmeasuring luminescence. Similar methods exist in which the environmentof the sample is converted to one favoring luciferase activity at thesame time that the luciferase enzyme is added. There are no ATPdetection systems that provide a composition or method capable ofinactivating endogenous ATPase activity and allowing for luciferaseactivity in the same environmental milieu. And there are no ATPdetection systems that provide a composition or method capable of lysingcells or extracting cellular ATP, inhibiting ATPase activity endogenousto a sample and allowing for luciferase activity in the sameenvironment. Therefore, current assays that use luminescence to detectATP are handicapped by the need for successive, time-consuming steps.

In preferred embodiments, the present invention reduces to a single stepthe manipulations needed for ATP detection in a sample, prior toluminescence measurement. In the single-step ATP assay of the invention,all of the necessary components of the ATP-dependent enzyme (e.g.,luciferase), such as the enzyme, substrates, and ATPase inhibitors arecomprised within a reagent composition and are added to a sample atonce. In some embodiments the reagent composition further comprises acell lysing agent or an agent for ATP extraction from cells. In someembodiments, a component of the reagent composition is a compound (e.g.,NaF) that prevents an increase in ATP levels in the sample over time.The mechanism by which ATP levels are increased over time in certainsamples, such as a cell lysate prepared with lymphoid cells (e.g.,Jurkat cells), is not well understood, but it possibly results from theactivity of a kinase enzyme endogenous to the sample. In someembodiments, a component of the reagent composition is an enzymestabilizing agent.

A. Definitions

Unless defined otherwise, all technical and scientific terms have thesame meaning as is commonly understood by one of skill in the art towhich this invention belongs. All cited patents and publications areincorporated by reference in their entirety unless otherwise noted.

The nomenclature recommendations of Demerec et al., 1966, where theseare relevant to genetics, are adapted herein. To distinguish betweengenes (and related nucleic acids) and the proteins that they encode, theabbreviations for genes are indicated by italicized (or underlined) textwhile abbreviations for the proteins start with a capital letter and arenot italicized. Thus, luc or luc refers to the luciferase nucleotidesequence that encodes luciferase polypeptide or Luc.

An “isolated” or “purified” luciferase is one that has been identifiedand separated and/or recovered from a component of its naturalenvironment.

The term “sample” as used herein, is used in its broadest sense. Asample is a composition suspected of containing ATP that is analyzedusing the invention. While often a sample is known to contain orsuspected of containing a cell or a population of cells, optionally in agrowth media, or a cell lysate, a sample may also be a solid surface,(e.g., a swab, membrane, filter, particle), suspected of containing anattached cell or population of cells. It is contemplated that for such asolid sample, an aqueous sample is made by adding the solid to thereagent composition of the invention or to another aqueous solution towhich the reagent composition of the invention is added. Filtration isdesirable in some cases to generate a sample, e.g., in testing a liquidor gaseous sample by a process of the invention. Filtration is preferredwhen a sample is taken from a large volume of a dilute gas or liquid.

The term “detection,” as used herein, refers to quantitatively orqualitatively determining the presence or absence of a component withinthe sample.

“Percent (%) amino acid sequence identity” is defined as the percentageof amino acid residues in one sequence that are identical to, with, oragainst amino acid residues in a second sequence in the region ofoverlap when the two sequences are optimally aligned. To determinepercent amino acid identity, sequences are locally aligned and ifnecessary, gaps are introduced to achieve the maximum percent sequenceidentity; conservative substitutions are not counted when calculatingsequence identity. Amino acid sequence alignment procedures to determinepercent identity are well known to those of skill in the art. Publiclyavailable computer software such as BLAST software (NCBI atwww.ncbi.nlm.nih.gov/BLAST/) may be used to align peptide sequences.Those skilled in the art can determine appropriate algorithms andparameters for measuring alignment, including any algorithms andparameters needed to achieve optimal alignment of two amino acidsequences.

When amino acid sequences are aligned, the percent amino acid sequenceidentity of a given amino acid sequence A to, with, or against a givenamino acid sequence B (which can alternatively be phrased as a givenamino acid sequence A that has or comprises a certain percent amino acidsequence identity to, with, or against a given amino acid sequence B)can be calculated as:% amino acid sequence identity=(X/Y)·100where X is the number of amino acid residues scored as identical matchesin the optimal alignment of A and B by the sequence alignment program oralgorithm and Y is the total number of amino acid positions aligned.B. Reagent Composition

The reagent composition of the present invention comprises one or moreATPase inhibitors, preferably a detergent, and a non-endogenousATP-dependent enzyme, where the composition is capable of maintaining atleast about 30% enzymatic activity for at least about one hour,preferably at least about 2 hours, more preferably at least about 4hours, compared to its activity just after (0 to 10 minutes) the enzymeis combined with the ATPase inhibitor, and where the one or more ATPaseinhibitors are present in the composition at a concentration sufficientto collectively reduce ATPase activity endogenous to the sample by atleast about 25%, more preferably at least about 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 99% or any increment therein relative to the ATPaseactivity endogenous to the sample in the absence of the ATPaseinhibitor. In preferred embodiments of the invention, the non-endogenousATP-dependent enzymes are luciferases.

1. Luciferases

Luciferase enzymes whose catalytic products include light, offersensitivity, a detectable product, and enable easy measurement of ATP.However, any luminescence-producing enzyme that is ATP-dependent may beused in the methods and compositions of the present invention.

At their most basic level, luciferases are defined by their ability toproduce luminescence. More specifically, a luciferase is an enzyme thatcatalyzes the oxidation of a substrate, luciferin, thereby producingoxiluciferin and photons.

To date, five classes of luciferases have been identified (Jones et al.,1999; Thomson et al., 1997). Of these, beetle luciferases, such as thatof the common firefly (family Lampyridae), form a distinct class withunique evolutionary origins (McElroy et al., 1969; White et al., 1969;White et al., 1975). Beetle luciferases are often referred to as fireflyluciferases in the literature; however, firefly luciferases are actuallya subgroup of the beetle luciferase class. Beetle luciferases may bepurified from the lanterns of the beetles themselves or from proteinexpression systems well known in the art (Baldwin and Green, 2000; Benyand Dolivo, 1976; Branchini et al., 1980; Filippova et al., 1989).

Beetle luciferases, particularly firefly luciferase from the NorthAmerican firefly photinus pyralis, are well known in the art. The P.pyralis luciferase (LucPpy) consists of approximately 550 amino acids ofM, 61 kDa as calculated by the protein encoded by the nucleotidesequence of the gene. However, other firefly luciferases are desirable,such as Photuris pennsylvanica firely luciferase (LucPpe2; 545 aminoacid residues; GenBank 2190534, (Ye et al., 1997)). Mutant luciferasesderived from LucPpe2 (e.g., LucPpe2 m78 (also known as 78-0B10), SEQ IDNO.:1; LucPpe2 m90 (also known as 90-1B5), SEQ ID NO.:2; LucPpe2 m133(also known as 133-1B2), SEQ ID NO.:3; LucPpe2 m146 (also known as146-1H2), SEQ ID NO.:4) are preferred; however, any luciferase thatmeets the limitations set forth herein may be used in the composition,method and kits of the invention. The method of making LucPpe2 m78,LucPpe2 m90, LucPpe2 m133, and LucPpe2 m146 is disclosed inPCT/US99/30925.

Isolated and/or purified luciferases are typically used in the presentinvention. Contaminant components of its natural environment arematerials that would typically interfere with diagnostic or therapeuticuses for the luciferase, and may include enzymes, hormones, and otherproteinaceous or non-proteinaceous materials. One technique to ascertainpurity is applying SDS-PAGE analysis under non-reducing or reducingconditions using Coomassie blue or silver stain. Isolated luciferaseincludes luciferase in situ within recombinant cells, since at least onecomponent of the luciferase natural environment will not be present.Luciferases can be isolated from biological specimens that produceluciferase or from a cell that expresses an exogenous polynucleotideencoding a desired luciferase (e.g., a nucleotide encoding 78-0B10,90-1B5, 133-1B2, or 146-1H2 (SEQ ID NOs.: 5-8, respectively)). Suchtechniques are well known to those of skill in the art.

The naturally-occurring substrate for beetle luciferases is fireflyluciferin, a polytherocyclic organic acid,D-(−)-2-(6′-hydroxy-2′-benzothiazolyl)-Δ²-thiazolin-4-carboxylic acid(luciferin). Luciferin may be isolated from nature (e.g. from fireflies)or synthesized. Synthetic luciferin can have the same structure as thenaturally occurring luciferin or can be derivatized, so long as itfunctions analogously (Bowie et al., 1973; Branchini, 2000; Craig etal., 1991; Miska and Geiger, 1987; Yang and Thomason, 1993). Examples ofderivatives of luciferin include D-luciferin methyl ester,D-luciferyl-L-phenylalanine, D-luciferyl-L-Nα-arginine,D-luciferin-O-sulphate and D-luciferin-O-phosphate (Miska and Geiger,1987), esters of luciferases that are hydrolyzed or acted upon byesterases to luciferin by components in a sample (Craig et al., 1991;Yang and Thomason, 1993). Other examples of useful luciferin analogsinclude naphthyl- and quinolylluciferin, which emit light in the greenand red light spectra respectively (Branchini et al., 1989). There aremultiple commercial sources for luciferin (e.g., Promega Corp., Madison,Wis.; Molecular Probes, Eugene, Oreg.).

The beetle luciferase-catalyzed reaction that yields luminescence (theluciferase-luciferin reaction) involves firefly luciferin, adenosinetriphosphate (ATP), magnesium, and molecular oxygen. In the initialreaction, the firefly luciferin and ATP react to form luciferyladenylate with the elimination of inorganic pyrophosphate. The luciferyladenylate remains tightly bound to the catalytic site of luciferase.When this form of the enzyme is exposed to molecular oxygen, theenzyme-bound luciferyl adenylate is oxidized to yield oxyluciferin in anelectronically excited state. The excited oxidized luciferin emits lighton returning to the ground state:

It is contemplated that the ATP function of the reaction can beperformed by an ATP analogue (e.g., dATP). It is also contemplated thatother ions can serve as substitutes for magnesium ions (e.g., Mn²⁺ orCa²⁺). Additionally, oxygen is a reactant of the reaction. Therefore,the reaction should not be conducted under anaerobic conditions.However, it is not generally necessary in practicing the invention toprovide oxygen over and above that present in the air. Reactions cantake place in closed vessels, provided there is sufficient oxygen in thereaction solution.

Most luciferase-luciferin reactions generate a flash of light that isshort-lived. However, some of the luciferases preferred for use with theinvention, e.g., LucPpe2 m146 and LucPpe2 m90 luciferases, under theconditions of the invention generate a “glow-type” luminescent signalwith less than 50% loss of luminescence per hour after the reagentcomposition is combined with the sample.

Any luciferase, luciferase variant, luciferase fragment, or variantluciferase fragment that retains the ability to generate luminescencewhen used in the reagent composition of the present invention and doesnot prevent the reagent composition from meeting the stabilityrequirements of the present invention, can be used in the presentinvention.

A full length luciferase variant will have at least about 80% amino acidsequence identity, preferably at least about 81% amino acid sequenceidentity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acidsequence identity and most preferably at least about 99% amino acidsequence identity with a full-length native luciferase sequence andretain the ability to generate luminescence. Ordinarily, variantluciferase fragments are at least about 50 amino acids in length, oftenat least about 60 amino acids in length, more often at least about 70,80, 90, 100, 150, 200, 300, 400, 500 or 550 amino acids in length, ormore, and retain the ability to generate luminescence. A luciferase,luciferase fragment, luciferase variant or variant luciferase fragmentmay be fused to other non-luciferase amino acid sequences and still befunctional in the invention.

Full length beetle luciferase, fragments of beetle luciferase, variantsof beetle luciferase, and variant fragments of beetle luciferase enzymeused in the compositions and methods of the present invention may bepurified from a native source or prepared by a number of techniques,including (1) chemical synthesis, (2) enzymatic (protease) digestion ofluciferase, and (3) recombinant DNA methods. Chemical synthesis methodsare well known in the art, as are methods that employ proteases tocleave specific sites. To produce segments of luciferase protein,segments of luciferase or luciferase variants can be made and thenexpressed in a host organism, such as E. coli. Methods such asendonuclease digestion or polymerase chain reaction (PCR) allow one ofskill in the art to generate an unlimited supply of well-definedfragments. Preferably, luciferase fragments share at least onebiological activity with native luciferase, as well as catalyticactivity, although the level of activity may vary from that of thenative luciferase.

Any type of amino acid substitution, insertion or deletion, orcombination thereof may be used to generate a variant luciferase.However, a luciferase with a conservative amino acid substitution ismore likely to retain activity. Useful conservative substitutions areshown in Table A “Preferred substitutions.” Conservative substitutionswhereby an amino acid of one class is replaced with another amino acidof the same type fall within the scope of the invention if thesubstitution does not impair luciferase activity. TABLE A Preferredsubstitutions Original Exemplary Preferred residue substitutionssubstitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn(N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) AsnAsn Glu (E) Asp Asp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg ArgIle (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, Ile,Val, Ile Met, Ala, Phe Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, IleLeu Phe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val(V) Ile, Leu, Met, Phe, Leu Ala, Norleucine

Non-conservative substitutions that effect (1) the structure of thepolypeptide backbone, such as a β-sheet or α-helical conformation, (2)the charge or (3) hydrophobicity, or (4) the bulk of the side chain ofthe target site might modify luciferase function. Residues are dividedinto groups based on common side-chain properties as denoted in Table B.Non-conservative substitutions entail exchanging a member of one ofthese classes for another class. TABLE B Amino acid classes Class Aminoacids Hydrophobic Norleucine, Met, Ala, Val, Leu, Ile neutralhydrophilic Cys, Ser, Thr Acidic Asp, Glu Basic Asn, Gln, His, Lys, Argdisrupt chain conformation Gly, Pro Aromatic Trp, Tyr, Phe

Variant luciferase genes or gene fragments can be made using methodsknown in the art such as oligonucleotide-mediated (site-directed)mutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis (Carter, 1986; Zoller and Smith, 1987), cassettemutagenesis, restriction selection mutagenesis (Wells et al., 1985) orother known techniques can be performed on the cloned DNA to produce theluciferase variant DNA (Ausubel et al., 1987; Sambrook, 1989).

2. Preferred Luciferases

Preferred luciferases of the invention possess catalytic activity thatdepends on ATP and emits photons. Preferred luciferases of the inventionhave enhanced chemostability in the presence of ATPase inhibitorsrelative to the level of the P. pyralis luciferase (LucPpy)chemostability in the same reaction conditions. Preferred luciferasesused in the compositions and methods of the invention generate a stablesignal, i.e., they yield enhanced duration of luminescence in aluciferase reaction defined as a less than 50% loss of luminescence perhour relative to the luminescence at the time the luciferase reactionwas initiated. Preferred luciferases of the invention allow for multipleanalyses of a sample over time or analysis of many samples over time,one hour after the luciferase is combined with the ATPase inhibitor,more preferably two hours and most preferably four hours or more.Optionally, the luciferases used in the compositions and methods of theinvention have enhanced thermostability properties. An exemplifiedpreferred luciferase is LucPpe2 m146 (SEQ ID NO.:4). Additional examplesof enzymes useful in the invention include, but are not limited to,LucPpe2 m78 (SEQ ID NO.:1), LucPpe2 m90 (SEQ ID NO.:2), and LucPpe2 m133(SEQ ID NO.:3).

The exemplified luciferases, LucPpe2 m78 (SEQ ID NO.:1), LucPpe2 m90(SEQ ID NO.:2), LucPpe2 m133 (SEQ ID NO.:3) and LucPpe2 m146 (SEQ IDNO.:4) were generated from a mutant of P. pennsylvanica (T249M). Thenucleic acid sequence encoding this protein was subjected to mutagenicmethods including recursive mutagenesis followed by screens forthermostability, signal stability, and substrate binding and is fullydescribed by Wood and Hall (WO 9914336, 1999).

Chemostability

“Chemostable luciferases” as used herein, defines luciferases thatretain activity in the presence of compounds or conditions when thosecompounds or conditions typically inhibit ATPases and disrupt thefunction of non-chemostable luciferases such as LucPpy. Theabove-identified exemplary luciferases [(LucPpe2 m78 (SEQ ID NO.:1),LucPpe2 m90 (SEQ ID NO.:2), LucPpe2 m133 (SEQ ID NO.:3) and LucPpe2 m146(SEQ ID NO.:4)] were found herein to have enhanced chemostability toATPase inhibitors.

Thus, preferred luciferases include those which maintain at least about30% (preferably at least about 60%, 70%, 80%, 90%, 95%, 99%) enzymaticactivity as measured by luminescence at least one hour (preferably atleast two hours, more preferably at least four hours) after contact withan amount of ATPase inhibitor, preferably a detergent, e.g., cationicdetergent (preferably DTAB or BDDABr), anionic detergent (preferablydeoxycholate or SDS) or zwitterionic detergent (preferably sulfobetaine3-10) or combination thereof sufficient to collectively reduce ATPaseactivity endogenous to a sample by at least about 25% (preferably atleast about 30%, even more preferably at least about 40%, 50%, 60%, 70%,80%, 90%, 95%, 99% or any increment therein) relative to the sample'sATPase activity in the absence of the ATPase inhibitor.

The chemostability of an enzyme also may be indicated by the rate ofdecline of its activity over time. For example, shortly (0 to 10minutes) after mixing the ATPase inhibitor and the luciferase, therebycreating the reagent composition, at several subsequent timepoints analiquot of the reagent composition is added to a sample and relativelight unit (RLU) measurements are obtained shortly thereafter. Thesemeasurements may be graphed to determine a trend of decline in enzymeactivity in the reagent composition over time.

The preferred chemostable luciferases (e.g., Ppe2 m78, Ppe2 m90, Ppe2m133, and Ppe2 m146) also retain activity in multi-detergent solutions.Specifically, solutions containing 0.01%, preferably 0.05%, 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, most preferably 0.25% CHAPS(3-([3-Cholamidopropyl]dimethylammonio)-1-propanesulfonate) with atleast 0.01%, preferably 0.05%, 0.1%, 0.2%, and most preferably 0.3% or1.0% BDDABr, taurocholic or taurolithocholic acids, or DTAB, or 0.01%,preferably 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,most preferably 1.0% of taurocholic or taurolithocholic acids with atleast 0.01%, preferably 0.05%, 0.1%, 0.2%, and most preferably 0.3% or1.0% BDDABr, DTAB, or CHAPS. Other multi-detergent solutions in whichLucPpe2 m78, LucPpe2 m90, LucPpe2 m133 and LucPpe2 m146 retain activityinclude 0.01%, preferably 0.05%, most preferably 0.1% TRITON X-100 withat least 0.01%, preferably 0.05%, 0.1%, 0.2%, 0.5%, most preferably 1.0%BDDABr, DTAB, or CHAPS; or 0.01%, preferably 0.05%, 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, most preferably 1.0% of taurocholicor taurolithocholic acids with at least 0.01%, preferably 0.05%, 0.1%,0.2% and most preferably 0.3 or 1.0% BDDABr, DTAB, or CHAPS; or 0.05%,1.0%, 2.0%, 4.0%, preferably 2% polyethylene glycol 400 dodecyl ether(THESIT®), with at least 0.05%, preferably 0.1%, 0.2% and mostpreferably 0.3% or 1.0% BDDABr, DTAB, or CHAPS.

Thermostability

In some embodiments, a thermostable luciferase that producesluminescence or other thermostable ATP-dependent enzyme that produces adetectable signal is desirable, especially in samples that are treatedwith heat immediately prior to ATP detection. A thermostable polypeptideremains active at temperatures that inactivate or denature otherproteins. The LucPpe2 m78, LucPpe2 m90, LucPpe2 m133 and LucPpe2 m146enzymes display increased thermostability compared to luciferases foundin nature or encoded from polynucleotides isolated from nature.

Signal Stability

Preferred luciferases used in the compositions and methods of theinvention generate a stable signal, i.e., such luciferases, when used ina luciferase reaction, yield luminescence with enhanced duration definedas less than 50% loss of luminescence per hour relative to theluminescence at the time the luciferase reaction was initiated. Thisproperty is referred to as signal stability. Preferred luciferases ofthe invention allow for multiple analyses of a sample over time oranalysis of many samples over time, at least one hour after theluciferase is combined with the ATPase inhibitor, more preferably atleast two hours and most preferably at least four hours or more. Thecombination of a luciferase and an ATPase inhibitor in the reagentcomposition, where the luciferase is capable of producing luminescencewith enhanced duration while in the presence of an ATPase inhibitor(and, optionally, kinase inhibitors) that stabilizes the amount of ATPpresent in the sample results in a reliable and efficient method fordetecting and quantifying cellular ATP for extended periods of time.

3. Other Desirable Luciferases

Any luciferase, luciferase fragment, or variants thereof that, in anATP-dependent manner, emits photons upon oxidation of a substrate and ischemostable, i.e., retains activity in the presence of the ATPaseinhibitors of the invention, may be used in the present invention. Otherdesirable characteristics, although not obligatory, such asthermostability and signal stability, are contemplated. In addition, theluciferase may be fused to another amino acid sequence and still befunctional in the present invenition. Such enzymes may be synthesized invitro or isolated from other organisms.

Other luciferases are found in bacteria, unicellular algae,coelenterates, beetles (other than P. pennsylvanica), fish, and otherorganisms. Chemically, all luciferases involve exergonic reactions ofmolecular oxygen with different luciferins, resulting in photonproduction (Hastings, 1996; Hastings and Wilson, 1976; Wilson andHastings, 1998; Wood et al., 1989). Preferably, other luciferases shouldbe dependent on ATP for oxidation of luciferin, or the reactionconditions manipulated such that bioluminescence generation depends onATP. One of skill in the art can ascertain ATP dependence for theluciferase-luciferin reaction.

The use of a luciferase other than that from beetles requires anappropriate luciferin molecule that upon oxidation generates achemically and electrically unstable intermediate or a detectableenzymatic product. Other substrates may be used, as well as otherATP-dependent enzymes that produce a detectable enzymatic product.Detectable products include photons, radioactively-labeled products,insoluble or soluble chromogens, or other products that can be detectedvisually or through the use of devices.

C. Kits

When the invention is supplied as a kit, the different components of thecomposition may be packaged in separate containers and admixed prior touse. Such separate packaging of the components permits long-term storagewithout loss of luciferase-luciferin activity. However, when the variousparts of the kit are admixed, thereby forming the “reagent composition,”the reagent composition comprises a luciferase, such as exemplified by,but not limited to, SEQ ID NOs.:1-4, and one or more ATPase inhibitorswhere the activity of the reagent composition has enhanced stability[i.e., the reagent composition is capable of maintaining at least about30%, more preferably at least about 60% activity for at least one hour,even more preferably at least 70%, 80%, 90%, 95%, 99% or greateractivity for at least one hour, still more preferably for at least twohours and even more preferably for at least four hours (as measured byluminescence when the reagent composition is combined with a sample)relative to the reagent composition's activity when it is first created,i.e., 0 to 10 minutes after the luciferase enzyme is first combined withan ATPase inhibitor], and where the ATPase inhibitor is present in thereagent composition at a concentration sufficient to reduce ATPaseactivity endogenous to a sample by at least about 25%, more preferablyat least about 30%, even more preferably at least about 40%, 50%, 60%,70%, 80%, 90%, 95%, or 99% or greater relative to the ATPase activity inthe absence of the ATPase inhibitor. Instructional materials may also beenclosed in the kit, as well as materials that may act as standards orcontrols, depending on the purpose of the kit.

1. The Reagent Composition

In a preferred embodiment, the components of the reagent composition ofthe invention can be supplied as two parts that are admixed shortlybefore use: (1) a part comprising luciferase and (2) a part comprisingone or more ATPase inhibitors. An example of such an embodiment isrepresented in Table C and others are represented in the Examples. Theluciferase component may further comprise luciferin and preferably islyophilized. The luciferase component optionally comprises excipientsfor lyophilization, protein (luciferase) stabilizer, magnesium (oralternative cation), and a magnesium chelator (or alternative cationchelator). The ATPase inhibitor component may further comprise a buffer,divalent cation metal chelators, magnesium (or alternative cation), adefoaming agent, anti-ATP-generating enzyme agents (e.g., NaF), anenzyme stabilizer (e.g., THESIT®) and cell lysing agent or agent forextracting ATP from cells. The different components of the invention maycomprise subsets of these parts and may be combined in any way thateither facilitates the application of the invention or prolongs storagelife. TABLE C Preferred components of a kit Component Action Preferredembodiments Luciferase/ Catalyzes luciferase- Ppe2m90 or Ppe2m146luciferin luciferin reaction luciferase in one step Substrate LuciferinLyophilization Highly purified porcine excipient and dermal collagen(Prionex) protein stabilizer Enzyme cofactor MgSO₄ Chelates Mg after1,2- ATP removal Cyclohexanediaminetetraacetic acid (CDTA) ATPase/Buffer Citrate buffer Extraction Potassium Phosphate buffer Buffer 2-(N-Morpholino)ethanesulfonic acid (MES) Chelates divalentEthylenediaminetetraacetic metal cations (EDTA) Defoamer MAZU DF204ATPase inhibitor DTAB Inhibitor of ATP- NaF generating activityNon-ionic detergent, THESIT ®, disrupts cellular Polyoxyethylene(9)-membranes lauryl-ether2. Luciferase-Luciferin Component

All luciferases, luciferase variants, luciferase fragments and variantluciferase fragments that catalyze an ATP-dependent reaction andgenerate luminescence are contemplated for use in the invention. Someembodiments eliminate the luciferin; for example, allowing a user tosupply a luciferin of his/her choice, or the luciferin may be providedseparately. The type of luciferin provided may vary but it must be asubstrate for the type of luciferase provided.

In one embodiment, a kit supplies the luciferase as an anhydrouspreparation. Anhydrous preparations of luciferase may be lyophilized, inwhich water is removed under vacuum, freeze-dried, crystallized, or anyother method that removes water that does not inactivate luciferase.Excipients that bulk the preparation and stabilize luciferase, such asserum albumins or Prionex, may also be included. In other embodiments,luciferase may be suspended in an aqueous composition comprisingglycerol or other solvent in which the enzyme is stable. The skilledartisan can easily determine the amounts of the various constituentsthat work in the compositions and methods of the invention.

3. ATPase Inhibitor Component

In a preferred embodiment, the kit comprises a component containing oneor more ATPase inhibitors within a solution optionally containing otherfunctional components, such as buffers, defoamers, enzyme stabilizers,and the like. This component may be supplied as a working solution or asa concentrate. A cell lysing agent or an agent that allows for cellularATP extraction (e.g., CTAB) may be packaged separately or together withthe ATPase inhibitor component. The ATPase inhibitor may be any of thosedescribed herein above. This component may further comprise agents thatchelate metal ions that may interfere with the luciferase-luciferinreaction (e.g. EDTA, EGTA), magnesium (preferably supplied as a salt,such as sulfate or chloride; or other functionally equivalent cation),defoaming agents, and inhibitors of ATP generating enzyme (e.g. NaF).Buffers that maintain pH of the working solution, e.g. citrate or MES(which may be supplied as a salt, such as sodium or free acid or base)or any other appropriate buffer may be used.

ATPase Inhibitor

One aspect of the invention is an ATPase inhibitor, preferably adetergent that inhibits ATPases, more preferably a detergent with acharged group, e.g., cationic detergent (preferably DTAB or BDDABr),anionic detergent (preferably deoxycholate or SDS) or zwitterionicdetergent (preferably sulfobetaine 3-10). Such inhibitors preventATPases endogenous to the sample from processing ATP to adenosinediphosphate (ADP) and adenosine monophosphate (AMP) before theluciferase is allowed to utilize the ATP in the sample for theluciferase-luciferin reaction. ATPase inhibitors may inactivate ATPasesdirectly or indirectly. They may bind to ATPases, either in the activesites, thus preventing substrate binding, or denature ATPases, such asby denaturing detergents, or they may selectively sequester ATPases fromtheir substrates.

One embodiment of the present invention uses cationic detergents such asDTAB or BDDABr detergents that act as ATPase inhibitors. However, otherATPase inhibitors are contemplated, such as other cationic detergents,anionic detergents (e.g., SDS and deoxycholate) and zwitterionicdetergents (e.g., sulfobetaine 3-10).

For DTAB or BDDABr the concentration in the reagent composition ispreferably in the range of about 0.02% to about 5.0%, more preferablyabout 0.05%, still more preferably about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% and 1.5% and mostpreferably to a final concentration of about 1.0% in the reagentcomposition.

Other non-cationic detergent ATPase inhibitors are contemplated forinclusion in the reagent composition; their requirements are that they,like DTAB, preferably inhibit at least about 25%, more preferably atleast about 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, still more preferably at least about 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% and most preferably about 100% ofendogenous ATPase activity in a sample when present in a reagentcomposition where the reagent composition is capable of maintaining atleast 30%, 40%, 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% and most preferably about 100% activity, as measuredby luminescence after the reagent composition is combined with thesample, for at least one hour, more preferably at least 2 hours comparedto the reagent composition's activity just after the luciferase iscombined with the ATPase inhibitor. Potentially suitable non-cationicdetergents that function as ATPase inhibitors include anionic detergents(preferably SDS and deoxycholate) and zwitterionic detergents(preferably sulfobetain 3-10). The concentration of a particular ATPaseinhibitor will vary depending on the inhibitor used, and to some extent,the sample being analyzed. One of skill in the art is familiar withmethods to determine the appropriate concentration of an ATPaseinhibitor for inclusion in the reagent composition; for example, theymay examine luciferin-luciferase derived signals over time, comparingthose samples that have varying concentrations of a candidate ATPaseinhibitor to those samples containing no known ATPase inhibitors.

It is fully anticipated that the most preferred concentration and eventhe concentration range functional in the methods of the invention willvary for different detergents. For example, SDS concentrationsfunctional in the methods of the invention are about 0.002% (Examples 2and 3). The functional concentration range for a detergent used in thepresent invention may readily be determined by one of skill in the artusing the methods disclosed herein.

It is contemplated that some ATPase inhibitors, at some of theconcentrations useful in the invention, may be insoluble or have lowsolubility in aqueous solutions. These compounds may first be dissolvedin an organic solution (e.g., dimethyl sulfoxide or dimethylformamide)and then diluted into the reagent composition for use in the compositionand methods of the invention.

Inhibitors of ATP-Generating Enzymes

In some samples, enzymes such as kinases may be active, allowing forcontinued production of ATP. Because the ATP concentration is determinedat a specific time, if such enzymatic activity is left unchecked, thenan overestimation of the ATP concentration will be made. To counter suchATP-generating activity, inhibitors of ATP production can be used.Although the action of a specific inhibitor may be incompletelyunderstood, their usefulness is not obviated. Examples of usefulcompounds include NaF, which is useful at concentrations of at least 1mM, preferably 2 mM to 100 mM or any increment therein; 2 mM is mostpreferred. Any such inhibitor may be used, however, if it does notadversely affect luciferase so as to take it outside the utility of theinvention. One of skill in the art will know how to determine theappropriate concentration of such an inhibitor, whether the inhibitor isnovel or well-known. Other inhibitors of ATP-generating enzymes include,but are not limited to, vanadate, paranitrophenylphosphate anddichloroacetic acid (Kiechle et al., 1980).

Buffers

Any buffers that maintain suitable pH for the working solution and donot interfere with the luciferase-luciferin reaction are contemplated.The preferred pH range is between about pH 4.5 and about pH 9.0, morepreferably between about pH 6.0 and about pH 8.0. In addition to MES andcitrate buffers, other buffers, such as phosphate buffered saline (PBS),Tris-N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), borate, and any otherbuffer known to those of skill in the art may be suitable. Selection ofappropriate buffers depends on pH buffering capacity and interactionwith the luciferase-luciferin reaction.

Defoamers

Defoaming agents are desirable to prevent foam from interfering with thedetection of bioluminescence, especially in applications that quantifyluminescence. Such agents as MAZU may be organic or silicone based.Selection of defoamers depends on their ability to eliminate foamwithout interfering with the luciferase-luciferin reaction.

Magnesium

The beetle luciferase-luciferin reaction is dependent not only on ATP,but also on magnesium ions. To ensure luciferase activity, magnesium isexogenously supplied. In addition to magnesium sulfate, other salts ofmagnesium are contemplated, such as magnesium chloride, magnesiumgluconate, magnesium acetate, magnesium bromide, magnesium carbonate,etc. In any case, the magnesium complex must dissociate to make Mg²⁺ions available to the luciferase and not interfere with theluciferase-luciferin reaction. One of skill in the art is aware thatother cations may be functional in place of magnesium. These includecalcium and manganese.

In some applications, endogenous magnesium should be sufficient, inwhich cases exogenous magnesium could be eliminated.

Cell Lysing Agents and ATP Extraction Agents

To free any sequestered ATP within a cell and to lyse cells in a sample,cell-lysing agents, such as non-ionic detergents, may be included. Anycell lysing agent is contemplated including other non-ionic detergents,(such as from the Triton series) cationic, anionic and zwitterionicdetergents, bile salts, chaotropes, and any other agent that disruptscellular membranes, including bacterial toxins such as oxylysins.Alternatively any agent that allows for ATP extraction from a cell iscontemplated (such as CTAB). Agents that allow for ATP extraction from acell include detergents present at a concentration that puts holes inthe cell membrane, allowing for ATP within the cell to leach into thesurrounding media, but not present at such a concentration that producesa cell lysate.

Stablizing Agents

While resistant to the action of nonionic and low concentrations ofzwitterionic detergents (Simpson and Hammond, 1991), native fireflyluciferase is inactivated by cationic detergents, such as benzalkoniumchloride, benzethonium chloride, CTAB (cetyltrimethylammonium), DTAB(dodecyltrimethylammonium bromide), and methylbenzethonium chloride(Simpson and Hammond, 1991).

The stabilizing agent can be any compound that stabilizes the luciferasefrom degradation. Suitable stabilizing agents include proteins (such asbovine serum albumin or gelatin) or detergents (preferably non-ionicdetergents, most preferably THESIT®).

Other Agents

Other agents that may be included in a kit include substances that areknown to enhance the duration of luminescence resulting from aluciferase reaction, such as co-enzyme A (CoA), thiol reagents, such asdithiothreitol and β mercaptoethanol (Wood, U.S. Pat. No. 5,283,179,1994; Wood, U.S. Pat. No. 5,650,289, 1997), metal ion chelators such asEDTA to prolong the signal and protease inhibitors (Scheirer, U.S. Pat.No. 5,618,682, 1997; Scheirer, U.S. Pat. No. 5,866,348, 1999), or highconcentrations of salts (Van Lune and Trer Wiel, WO 00/18953, 2000).

Other Kit Contents

Kits may also include reagents in separate containers that facilitatethe execution of a specific test, such as cell viability, cytotoxicity,cell proliferation, or determination of ATP concentration. For example,ATP may be supplied so that standard curves may be determined or for useas internal controls. Substances that are known to be cytotoxic to cellscan be included for use as a positive control in tests of cell viabilityor for the effects of compounds on cells. The kit may supply a samplegathering component such as a membrane, filter or swab.

4. Containers or Vessels

The reagents included in the kits can be supplied in containers of anysort such that the life of the different components are preserved, andare not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized luciferase orbuffer that have been packaged under a neutral, non-reacting gas, suchas nitrogen. Ampules may consist of any suitable material, such asglass, organic polymers (such as polycarbonate, polystyrene, etc.,ceramic, metal or any other material typically employed to holdreagents. Other examples of suitable containers include simple bottlesthat may be fabricated from similar substances as ampules, andenvelopes, that may have interiors lined with foil, such as aluminum oran alloy. Other containers include test tubes, vials, flasks, bottles,syringes, or the like. Containers may have a sterile access port, suchas a bottle having a stopper that can be pierced by a hypodermicinjection needle. Other containers may have two compartments that areseparated by a readily removable membrane that upon removal permits thecomponents to mix. Removable membranes may be glass, plastic, rubber,etc.

5. Instructional Materials

Kits may also be supplied with instructional materials. Instructions maybe printed on paper or other substrate, and/or may be supplied as anelectronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zipdisc, videotape, audio tape, etc. Detailed instructions may not bephysically associated with the kit; instead, a user may be directed toan internet web site specified by the manufacturer or distributor of thekit, or supplied as electronic mail. In a preferred embodiment, theinstructions instruct the user to combine the luciferase with the ATPaseinhibitor before adding the reagent composition to a sample.

D. Reagent Composition Activity

To measure luminescence and thereby determine the reagent compositionactivity, the relative light unit (RLU) value generated by theluciferase reaction at a timepoint of interest after the reagentcomposition is combined with a sample may be measured. For example, anRLU value may be obtained by measuring the resulting luminescence from asample with a known concentration of ATP combined with the reagentcomposition just after (0-10 min) the component comprising the ATPaseinhibitor is added to the component comprising the luciferase therebycreating the reagent composition. This is considered 100% activity (timezero) under those conditions. If, after combining the componentcomprising the ATPase inhibitor with the component comprising theluciferase and thereby generating the reagent composition, the reagentcomposition is left for two hours, preferably in the temperature rangeof room temperature (about 20° C.-about 25° C.) to about 37° C., priorto measuring luminescence under identical conditions as the time 0assay, and the RLU value obtained is greater than 60% of that obtainedat time 0, then the reagent composition retained at least 60% of itsactivity for two hours.

A reagent composition of the present invention retains 30%, 40%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or anyincrement therein and most preferably 100% of its activity, as measuredby luminescence after the reagent composition is combined with thesample for at least one hour, preferably for at least two hours,relative to its activity when formulated (time 0)—that is from the timethe component comprising the ATPase inhibitor was added to the componentcomprising luciferase or shortly thereafter (0-10 minutes).

In one preferred embodiment, the working stock of the reagentcomposition comprises DTAB or BDDBr in concentrations of about 0.02%(preferably about 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%,0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%,2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%,9.5%, 10% and any increment therein, more preferably about 1%) andretains at least about 30% (preferably at least about 40%, 50%, 60%,70%, 80%, 90%, 95%, 99%) of activity at least one hour (preferably atleast two hours) after formulation.

In another preferred embodiment, the reagent compositions comprisesulfobetaine at a concentration of 0.6%, 0.7%, 0.8%, 0.9% or 1.0% or anyincrement therein, SDS at a concentration of 0.001%, 0.002%, 0.003%,0.004% or 0.005% or any increment therein, or deoxycholate at aconcentration of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% or any incrementtherein and retain at least about 30% (preferably at least about 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%) of activity at least one hour(preferably at least two hours) after formulation.

E. Detecting and Quantifying the Products of the Luciferase-LuciferinReaction

A beetle luciferase-luciferin reaction results in the generation oflight (“luminescence”). The invention provides assays for ATPmeasurement by measuring luminescence. Users may simply visually inspectsample reactions to ascertain the production of light. However, moresensitive instrumentations allow not only detection of faint signals,but also quantification of the light signal. Also contemplated arereactions in which non-light products are measured, according to thenature of the products. Any assay for measurement of ATP that results ina signal may benefit from the present invention. Appropriate instrumentsand methods for such products will be apparent to the skilled artisan.

In all cases in which light is detected, specialized instruments, suchas luminometers, can read the light product of a luciferase-luciferinreaction. Any instrument that can detect the light of the wavelengthsemitted by the luciferase-luciferin reaction may be used. Suchinstruments may read samples singularly, or, in high-throughput screens,may read many samples housed in the wells of microwell plates (6, 24,48, 96, 384, 1536 and so on, well formats). Clearly, the devices used tomeasure the emitted light do not limit the invention. Other devices thatcan be used include scintillation counters (Nguyen et al., 1988) orinstruments invented or adapted to be sensitive to luminescence, such asphotometers (Picciolo et al., 1977). Photographic film or X-ray film mayalso be used to detect luminescence. In addition, a user may visuallyinspect a sample to qualitatively evaluate luminescence.

F. Uses for ATP-Dependent Luciferase-Luciferin Reactions

Because the beetle luciferase-luciferin reaction is ATP-dependent,luciferase can be used to assay for ATP. The reaction is remarkablysensitive, allowing ATP to be detected in a sample containing as littleas 10-16 moles ATP or less. This sensitivity can be exploited tounderstand cell viability and the effects that exogenous substances mayexert on cell metabolism and viability. In a cellular context, ATPpowers cellular metabolism, the presence of ATP correlates to anactively metabolizing cell, i.e. the cell is “viable.”

The invention is drawn to methods, compositions and kits that are usedto effectively and accurately detect and quantify cellular ATP levels,exploiting the ATP-dependence of beetle luciferase to oxidize luciferin.

The invention comprises the addition of a single composition (reagentcomposition) that comprises a luciferase and at least one ATPaseinhibitor to a sample and the detection of luminescence. Optionally, akinase inhibitor or a compound that prevents accumulation of ATP canalso be present in the reagent composition. Additionally, a cell-lysingagent (e.g., a polyoxyethylene such as THESIT®) or an ATP extractingagent may be present in the composition. This single step comprisingadding the reagent composition followed by reading the luminescencerepresents a significant advance in assays for ATP.

1. Detecting ATP

The methods, compositions and kits of the invention provide for thesimple qualitative or quantitative detection of ATP (or ATP analoguewhich can function as a luciferase substrate) in a sample. In preferredembodiments, a simple qualitative experiment in which luminescence isgenerated in a sample using the invention, indicates the presence ofATP. Luminescence is generated using a reagent composition comprisingluciferase such as LucPpe2 m78, LucPpe2 m90, LucPpe2 m133 or LucPpe2m146, and one or more ATPase inhibitors. In addition, the reagentcomposition may further comprise one or more of the followingcomponents: luciferin, which may be reconstituted from a lyophilizedpreparation, (alternatively, an appropriate luciferin-analoguesubstrate), ATPase inhibitor(s), inhibitor(s) of ATP-generating enzymessuch as kinases, divalent cation (e.g. magnesium), enzyme stabilizingagent, buffer, cell-lysis agent or cellular ATP extracting agent.

A sample may be anything that is suspected of containing ATP or ATPanalogue, such as cell lysates, intact cells, biopsies, foods,beverages, swabs wiped on surfaces such as those of animals, plants, orinanimate objects, and the like. Other examples of samples includecompositions of a known ATP concentration. Cells or cell lysates may befrom any organism, prokaryotic or eukaryotic. Examples of prokaryoticcells include E. coli, P. aeruginosa, B. subtilis, and S. typhimurium.Eukaryotic cells may be from plants, animals, fungi, insects, etc. orcultured cells from such organisms. Examples include A. thaliana andBrassica sp., Chlamydonmonas sp. and Volvox sp. (plants), H. sapiens andMus sp. (animals), Saccharoymyces sp. (esp. cerevisae and pombe) andNeurospora sp. (fungi), D. melanogaster and C. elegans (insects), invitro cultured callus cells from any plant, primary cells cultured invitro from any organism (such as organ explants from, for example,rodents), mammalian cell lines such as Madin-Darby canine kidney (MDCK)and Chinese hamster ovary (CHO) cells, and insect cell lines such as Zcells. These examples are furnished only as examples and are not meantto be limiting.

A cell lysate comprises cellular components that are no longer organizedinto a recognizable intact cellular architecture. Cell lysates may havesoluble and insoluble components, either of which may be removed beforeusing the lysate. Lysates may be prepared by any means, includingphysical disruption using sonication, a dounce, mortar and pestle,freeze-thaw cycling, or any other device or process that destroys thephysical integrity of cells; or lysis by detergents, such as those inwhich LucPpe2 m146 retains activity, such as zwitterionic and nonionicdetergents, or cationic detergents DTAB or CTAB. Preferably, the celllysate is produced in such a way that the integrity of the ATPconcentration is preserved at the time the cells are harvested. Toaccurately detect ATP in a sample, enzymes that would degrade cellularATP or those that would generate ATP are preferably inhibited. In theabsence of such inhibitors, an inaccurate determination of ATPconcentration risks being made. Inhibitors such as DTAB inactivateATPases, while other molecules such as NaF inactivate ATP-generatingenzyme activity. It is hypothesized, yet not fully understood, that forthose cell types in which NaF is effective (e.g., lymphoid cells), NaFis potentially acting to inhibit (a) kinase(s).

Inhibitors of ATP-generating enzymes, those enzymes that have as aproduct or by-product ATP, such as the activity of kinases, may beincorporated into the reagent composition. An example of an effectiveinhibitor is NaF (Bostick et al., 1982). Such compositions comprise NaFat concentrations of at least 0.5 mM, preferably at least 1 mM, morepreferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,953, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM or anyincrement therein; 2 mM is most preferred. Other inhibitors ofATP-generating enzymes include other kinase inhibitors, such asvanadate, AMP, DAPP (Bostick et al., 1982) and dichloroacetic acid(Kiechle et al., 1980).

2. Quantifying ATP

The compositions, methods and kits of the invention permit a user toquantify the amount of ATP in a sample by quantifying the amount ofluminescence. The invention is applied to a sample of interest, and alsoto samples containing known amounts of ATP (controls). The signalgenerated from applying the invention to a sample of unknown ATPconcentration is correlated to signals generated either by internalcontrols (the addition of a known amount of ATP to a sample andmeasuring the subsequent luminescence) or external standard curves,generated by measuring the luminescence of several samples of known ATPconcentrations and plotting them graphically. Such methods are known toskilled artisans. (Moyer and Henderson, 1983; Ronner et al., 1999;Stanley, 1989; Wood et al., 1989).

3. Cell Viability

The presence of ATP in a cell, eukaryotic or prokaryotic, indicatesactive metabolic processes, indicating a viable cell. The compositions,methods and kits of the present invention can be used to assay cellviability (Cree, 1998; Jassim et al., 1990; Petty et al., 1995). Anaccurate measure of cell viability allows for the accurate assessment ofthe effects of substances on cells; other purposes for determining cellviability are well-known to those of skill in the art.

Determining cell viability is useful, for example, to determinecytotoxicity, cell proliferation, biological phenomena, necrosis, oralterations in cellular metabolism. Cell viability assays can alsodetermine the overall viability of a cell population.

The sample in which ATP is measured to determine cell viability may beviable cells themselves, a cell lysate or any other sample suspected ofcontaining cells. When using cells, modified beetle luciferases that aremembrane permeable may be used (for example, see Craig et al., 1991). Inmany cases, however, a cell lysate is preferred.

4. Effects of Compounds on Cells

The compositions, methods and kits of the present invention can beapplied to measure the effects of compounds, such as inorganics, smallorganics, peptides, proteins and polypeptides, on cellular metabolismwhen contacted with a sample (Aiginger et al., 1980; Andreotti et al.,1995; Bradbury et al., 2000; Cree and Andreotti, 1997; Crouch et al.,1993; Kangas et al., 1984). Determining the effects of compounds oncells can assess the measure of a potential pharmaceutical composition'seffectiveness. Cytotoxic compounds—those that kill cells—can be usefulin the treatment of cancer cells, especially if they selectively killquickly-dividing cells. In other cases, a compound with some otherusefulness may be negated if a cytotoxic effect is not desired. BecauseATP is a measure of a cell's “metabolic” health, an abnormal surge ordepression of ATP reduction indicates a change in cellular homeostasis.Compounds that contact cells can influence ATP production through alarge number of mechanisms, most notably cell death and cellproliferation. These compounds may be catalogued in compound libraries,or tested singly. Such applications of the invention apply controls inwhich samples are contacted with control substances whose effects on ATPmetabolism are known. Also preferably, controls include samples in whichluciferase and the compound are present together to assure that thecompound itself is not directly affecting luciferase activity.

The following examples are intended to illustrate the present inventionwithout limitation.

EXAMPLES Example 1 I. Detergents that Inhibit ATPases

This example was designed to test the ability of different detergents toinhibit ATPase activity endogenous to cells and demonstrate the level ofsuch inhibition. Three separate detergents in each of four detergentclasses were tested: anionic [SDS (Sodium dodecyl sulfate), Bioterge (ana olefin sulfate), and sodium deoxycholate], nonionic [TRITON X-100,BigCHAP (N,N-bis(3-D-Gluconamidopropyl)cholamide) and THESIT®(polyethylene glycol 400 dodecyl ether, Fluka, #88315)], cationic[BDDABr (Benzyldimethyldodecylammonium bromide), CTAB(Cetyltrimethylammonium bromide), and DTAB (dodecyltrimethylammoniumbromide)] and zwitterionic [CHAPS(3[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid), CHAPSO(3-([3-Cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate),and Sulfobetaine 3-10(N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate)]. Detergents wereobtained from Sigma, Fluka and Aldrich.

L929 cells (ATCC CCL-1; 1.5×10⁵ cells/ml) in F12/DMEM (Hyclone, SH30023)containing 10% horse serum (Hyclone, SH30074) were frozen and thawed forfour cycles to create a cell lysate. Then 50 μl of stock detergentsolutions [10%, 5.0%, 1.0%, and 0.5% (w/v)] were separately added to 450μl cell lysate, creating a final detergent concentration in the celllysate sample of 1.0%, 0.5%, 0.1%, and 0.05%, respectively (thesepercentage values are those used in Tables D and E herein below).Control samples contained either lysate only or 1.0 μM ATP in 15 mMHEPES buffer (pH 7.5). All samples were incubated at 22° C. (roomtemperature) for the duration of the experiment.

At various timepoints, 20 μl from each sample/detergent mixture wasadded to a 96-well luminometer plate in triplicate, and then 100 μl of asolution (“luciferase-luciferin” or “L/L reagent”) containing 25 mMHEPES buffer (pH 7.5), 40 μg luciferase enzyme LucPpe2 m146 (Promega,E140), 100 μM luciferin (Promega), and 10 mM MgSO₄ was added to eachwell. The L/L reagent was stored at 4° C. for the duration of theexperiment and then allowed to reach ambient temperature just beforeassaying. After mixing the contents of the plate, light output wasmeasured with a Dynex MLX microtiter plate luminometer (Chantilly, Va.),0.5 second reads per well with the first measurement taken five minutesafter the detergent solution was combined with the sample. The averagerelative light units and the timepoint at which it was measured (RLU;Table D) and percent of remaining luciferase activity at the timepoint(Table E) are reported; controls were run with each set of experimentalconditions as indicated. The cationic and anionic detergents wereassayed on one day with one set of controls. The zwitterionic andnonionic detergents were assayed on a separate day with a separate setof controls. The DTAB at 0.5% was assayed on both days (1 and 2). If theoriginal average RLUs reported in Table D were below 5.0, the percent oforiginal activity values were not recorded in Table E as the detergentwas determined to be present at a concentration that significantlydestroyed luciferase activity. TABLE D Average Relative Light UnitValues 5 min 75 min 169 min 222 min 260 min Controls Cell lysate 6713.331844.71 953.35 384.77 200.78 (cationic and anionic) Cell lysate 6748.772297.65 1212.43 508.38 246.19 (nonionic and zwitterionic) ATP Control19986.2 16628.6 17439.9 20174.3 15668.7 (cationic and anionic) ATPControl 17779.6 18218.8 16639.6 18886.2 17952.2 (nonionic andzwitterionic) Anionic Detergents SDS 1.0% 0.01 0.00 0.01 0.00 0.01 0.5%0.00 0.00 0.02 0.00 0.00 0.1% 0.31 0.18 0.21 0.26 0.34 0.05% 1181.171060.03 949.7 921.86 803.0 Bioterge 1.0% 0.01 0.01 0.01 0.01 0.02 0.5%0.1 0.04 0.05 0.05 0.05 0.1% 4788.81 1445.91 167.09 96.43 64.83 0.05%5343.62 1004.34 435.52 196.51 107.54 Deoxycholate 1.0% 3.98 3.63 3.633.15 3.24 0.5% 3189.56 2715.45 2268.77 2105.38 1845.36 0.1% 6422.343730.44 1627.81 1028.42 669.31 0.05% 5810.85 1729.95 738.1 375.74 188.59Non-ionic Detergents TRITON X-100 1.0% 6073.78 1774.10 839.14 331.95174.89 0.5% 6106.61 1880.98 871.01 378.58 195.42 0.1% 6837.20 3004.571643.05 801.38 448.93 0.05% 6160.71 1964.17 822.48 303.73 147.48 BIGCHAP1.0% 7576.05 4043.35 2474.59 1374.29 822.65 0.5% 7438.53 3618.22 2092.691144.80 677.61 0.1% 6607.01 2087.01 1363.07 634.92 301.55 0.05% 6410.422260.45 1036.69 487.72 238.44 THESIT ® 1.0% 6204.02 1979.43 968.14416.45 210.78 0.5% 6392.75 2304.48 1122.24 487.28 240.72 0.1% 6022.322654.97 1509.07 755.94 447.44 0.05% 5632.96 1601.09 623.38 211.67 110.15Cationic Detergents BDDABr 1.0% 0.49 0.51 0.44 0.42 0.43 0.5% 122.47107.39 93.92 96.68 101.11 0.1% 7069.08 5136.92 3260.59 2366.49 1767.580.05% 7077.46 4539.97 2465.57 1680.16 1233.82 CTAB 1.0% 0.09 0.08 0.070.06 0.07 0.5% 0.34 0.28 0.23 0.27 0.18 0.1% 5104.82 3607.65 1889.291248.51 not done 0.05% 6525.51 4029.32 2093.80 1437.67 1002.72 DTAB 1.0%800.61 693.13 649.41 671.03 645.40 0.5% (day1) 5630.61 5100.09 4957.484916.46 4515.20 0.5% (day2) 6617.08 6341.09 5977.33 5824.18 5622.12 0.1%6991.01 4753.46 2737.36 1950.95 1408.18 0.05% 6487.93 3138.81 1496.01943.40 638.30 Zwitterionic Detergents CHAPS 1.0% 7241.33 3816.06 2348.281322.30 809.22 0.5% 7368.91 4048.24 2548.39 1438.57 909.15 0.1% 6515.772583.65 1236.53 448.96 284.18 0.05% 6356.62 2305.56 1143.02 500.80240.81 CHAPSO 1.0% 7160.63 3803.84 2376.31 1332.97 840.57 0.5% 7422.264089.32 2581.01 1460.54 923.81 0.1% 6549.85 2584.56 1210.14 558.69280.84 0.05% 6396.53 2424.54 1124.83 480.09 238.80 Sulfobetaine 3-101.0% 7861.36 5703.51 4318.46 3075.12 2193.02 0.5% 6565.98 2405.951211.13 609.75 391.64 0.1% 6506.61 2367.27 1100.37 446.29 222.22 0.05%6247.31 2270.13 1053.56 465.03 233.19

TABLE E Percent ATP Remaining Condition 5 min 75 min 169 min 222 min 260min Cell lysate (cationic 100 27.5 14.2 5.7 3.0 and anionic) Cell lysate100 34.0 18.0 7.5 3.6 (nonionic and zwitterionic) ATP Control 100 83.287.3 100.9 78.4 (cationic and anionic) ATP Control 100 102.5 93.6 106.2101.0 (nonionic and zwitterionic) Anionic Detergents SDS 0.05% 100 89.780.4 78.0 68.0 Bioterge 0.1% 100 30.2 3.5 2.0 1.4 0.05% 100 18.8 8.2 3.72.0 Deoxycholate 0.5% 100 85.1 71.1 66.0 57.9 0.1% 100 57.9 25.3 16.010.4 0.05% 100 29.8 12.7 6.5 3.2 Non-ionic Detergents TRITON X-100 1.0%100 29.2 13.8 5.5 2.9 0.5% 100 30.8 14.3 6.2 3.2 0.1% 100 43.9 24.0 11.76.6 0.05% 100 31.9 13.4 4.9 2.4 BIGCHAP 1.0% 100 53.4 32.7 18.1 10.90.5% 100 48.6 28.1 15.4 9.1 0.1% 100 31.6 20.6 9.6 4.6 0.05% 100 35.316.2 7.6 3.7 THESIT ® 1.0% 100 31.9 15.6 6.7 3.4 0.5% 100 36.0 17.6 7.63.8 0.1% 100 44.1 25.1 12.6 7.4 0.05% 100 28.4 11.1 3.8 2.0 CationicDetergents BDDABr 0.5% 100 87.7 76.2 78.9 82.6 0.1% 100 72.7 46.1 33.525.0 0.05% 100 64.1 34.8 23.7 17.4 CTAB 0.1% 100 70.7 37.0 24.5 not done0.05% 100 61.7 32.1 22.0 15.4 DTAB 1.0% 100 86.6 81.1 83.8 80.6 0.5% (1)100 90.6 88.0 87.3 80.2 0.5% (2) 100 95.8 90.3 88.0 85.0 0.1% 100 68.039.2 27.9 20.1 0.05% 100 48.4 23.1 14.5 9.8 Zwitterionic DetergentsCHAPS 1.0% 100 52.7 32.4 18.3 11.2 0.5% 100 54.9 34.6 19.5 12.3 0.1% 10039.7 19.0 8.6 4.4 0.05% 100 36.3 18.0 7.9 3.8 CHAPSO 1.0% 100 53.1 33.218.6 11.7 0.5% 100 55.1 34.8 19.7 12.4 0.1% 100 39.5 18.5 8.5 4.3 0.05%100 37.9 17.6 7.5 3.7 Sulfobetaine 3-10 1.0% 100 72.6 54.9 39.1 27.90.5% 100 36.6 18.4 9.3 6.0 0.1% 100 36.4 16.9 6.9 3.4 0.05% 100 36.316.9 7.4 3.7

The data demonstrate that for the anionic detergents tested, SDS atconcentration 0.05% and deoxycholate at concentrations 0.5% and 0.1%slow the degradation of endogenous ATP in the cell lysate resulting infrom about three to about twenty times more ATP present in the sampleafter four hours when the detergent was present than when it was absentindicating inhibition of ATPase endogenous to the sample. The non-ionicdetergents had little, if any, additional ATP present in the sampleafter four hours in the presence of the detergent than in the absence ofthe detergent indicating that these detergents did not inhibitendogenous ATPase. The zwitterionic detergents tested had three to fourtimes more ATP present in the sample after four hours when the detergentwas present than when it was absent. The most significant results wereseen when cationic detergents were incubated with the cell lysate.BDDABr at 0.5% concentration, and DTAB at 0.5% and 1.0% concentrations,each had at least 25-fold more ATP present in the sample after fourhours than samples without these detergents. CTAB at 0.05% concentrationhad about four times more ATP present in the sample after four hourswhen the detergent was present than when it was absent.

Example 2 II. Detergents That Inhibit ATPases

This example tested the seven detergents that demonstrated inhibition ofATPase activity in Example 1 herein above at a lower percentage than waspreviously tested. The experiment was performed as detailed inExample 1. The average relative light unit values and the percent of ATPremaining values are listed below in Table F and G respectively. TABLE FAverage Relative Light Unit Values 5 min 75 min 169 min 222 min 260 minControls Cell Lysate 5296.25 1613.68 334.28 225.95 123.08 ATP + Hepes22565.95 16238.00 20241.55 19302.50 18528.1 Samples 0.5% DTAB 5189.84564.65 4111.89 4230.51 4267.32 0.02% DTAB 5870.43 1997.09 435.05 329.83227.72 0.02% BDDABr 6215.32 2686.49 962.85 604.47 429.86 0.02%Sulfobetaine 6120.5 1622.61 291.09 245.55 136.43 0.02% Deoxycholate5962.61 1694.47 368.11 241.05 135.25 0.01% SDS 13116.20 10753.0312051.63 11822.03 11900.73 0.002% SDS 5773.39 1574.34 278.67 236.28128.58

TABLE G Percent ATP Remaining Condition 5 min 75 min 169 min 222 min 260min Lysate Control 100.00 30.47 6.31 4.27 2.32 ATP Control 100.00 71.9689.70 85.54 82.11 0.5% DTAB 100.00 87.95 79.23 81.52 82.23 0.02% DTAB100.00 34.02 7.41 5.62 3.88 0.02% CTAB 100.00 43.22 15.49 9.73 6.920.02% BDDABr 100.00 48.53 21.51 13.18 9.74 0.02% Sulfobetaine 100.0026.51 4.76 4.01 2.23 0.02% Deoxychol. 100.00 28.42 6.17 4.04 2.27 0.01%SDS 100.00 48.62 25.80 14.20 8.79 0.002% SDS 100.00 27.27 4.83 4.09 2.23

The log of the percent of ATP remaining after original timepoint valuesfrom Table E and Table G were plotted on the y axis against time (inminutes) on the x axis. The slope of the line generated by these valuesin the presence of detergent was divided by the slope of the linegenerated by the values in the absence of detergent (lysate control)resulting in the Relative ATPase Activity values listed below in TableH. TABLE H RELATIVE ATPase ACTIVITY slope det/ Relative ATPase ActivitySlope slope lysate Controls: Cell lysate (cat & an) −0.0056 1.000 Celllysate (zwit & non) −0.0053 1.000 ATP control (cat & an) −0.0002 0.030ATP control (zwit & non) 0.00002 −0.005 ATP control for detergents−0.00013 0.02 less than 0.05% Percent detergent Anionic: SDS 1.00 — —0.50 — — 0.10 — — 0.05 −0.0006 0.105 0.01 −0.00385 0.702 0.002 −0.00641.010 Bioterge 1.00 — — 0.50 — — 0.10 −0.0076 1.366 0.05 −0.0062 1.113Deoxycholate 1.00 — — 0.50 −0.0009 0.158 0.10 −0.0038 0.684 0.05 −0.00550.984 0.02 −0.0064 1.004 Non-ionic: Triton X-100 1.00 −0.0061 1.152 0.50−0.0059 1.117 0.10 −0.0047 0.889 0.05 −0.0065 1.228 BIGCHAP 1.00 −0.00380.725 0.50 −0.0041 0.782 0.10 −0.0051 0.968 0.05 −0.0056 1.069 THESIT ®1.00 −0.0058 1.096 0.50 −0.0056 1.065 0.10 −0.0045 0.850 0.05 −0.00681.295 Cationic: BDDABr 1.00 — — 0.50 −0.0004 0.064 0.10 −0.0023 0.4140.05 −0.0030 0.528 0.02 −0.0039 0.623 CTAB 1.00 — — 0.50 — — 0.10−0.0028 0.507 0.05 −0.0031 0.561 0.02 −0.0046 0.718 DTAB 1.00 −0.00030.057 0.50 −0.0003 0.055 0.50 −0.0003 0.048 0.10 −0.0027 0.480 0.05−0.0039 0.690 0.02 −0.0056 0.884 Zwitterionic: CHAPS 1.00 −0.0038 0.7150.50 −0.0036 0.688 0.10 −0.0054 1.023 0.05 −0.0056 1.061 CHAPSO 1.00−0.0037 0.703 0.50 −0.0036 0.685 0.10 −0.0054 1.030 0.05 −0.0057 1.080Sulfobetaine 3-10 1.00 −0.0022 0.417 0.50 −0.0049 0.921 0.10 −0.00581.109 0.05 −0.0057 1.077 0.02 −0.0064 1.009

A relative activity value of 1.0 or greater indicates 100% cellularATPase activity at the concentration of detergent tested. A relativeactivity value of 0.5 indicates a two-fold (or 50%) decrease in thelevel of cellular ATPase activity at the concentration of detergenttested when compared to the ATPase activity level in the absence of thedetergent. A relative activity value of 0.2 indicates a five-fold (or80%) decrease in the level of cellular ATPase activity at theconcentration of detergent tested when compared to the ATPase activitylevel in the absence of the detergent.

For the cationic detergents tested, the reaction conditions whichresulted in a 25% or greater decrease in the relative ATPase activitywere DTAB at concentrations of 0.05% and greater; CTAB at concentrationsof 0.02% and greater; BDDABr at concentrations of 0.02% and greater.Therefore, all cationic detergents tested in this assay at aconcentration of 0.05% or greater decreased cellular ATPase activity by25% or more. For the anionic detergents tested, the reaction conditionswhich resulted in a 25% or greater decrease in relative ATPase activitycompared to reactions in the absence of detergent were SDS atconcentrations of 0.01% and greater and deoxycholate at concentrationsof 0.1% and greater. None of the nonionic detergents tested resulted ina 25% or greater decrease in the level of cellular ATPase activity. Forthe zwitterionic detergents tested, only sulfobetaine at a concentrationof 1% or greater was able to decrease the relative ATPase activity by25% or greater compared to reactions in the absence of detergent.

These values are graphed in FIG. 1 (nonionic and zwitterionicdetergents) and FIG. 2 (cationic and anionic detergents).

Example 3 Stability of Reagent Composition (Room Temp.)

The detergents used for inhibition of endogenous ATPase activity in asample also affect activity of luciferase. This example was designed totest the stability and functionality of the reagent composition(“reagent composition”) over time when it comprised different luciferaseenzymes and when the ATPase inhibitor was present at a concentrationthat significantly inhibited the ATPase activity endogenous to thesample as demonstrated in Example 1 and Example 2 herein above. It isultimately the extended stability of a reagent composition, comprisingan ATPase inhibitor and a luciferase enzyme, which provides acomposition useful for measuring ATP, in a sample or samples, over anextended period of time. The most preferred luciferase for use in such areagent composition would be one whose stability is minimally decreasedin the presence of an ATPase inhibitor when that ATPase inhibitor ispresent in the reagent composition at a concentration capable ofinhibiting at least 30% ATPase activity endogenous to the sample.

In this experiment, the stability of a reagent composition comprisingwild type LucPpy was compared to the stability of a reagent compositioncomprising LucPpe2 m146 at about 23° C. (room temperature). The variousreagent compositions had varying detergent concentrations. The variousdetergents tested were those demonstrated in Examples 1 and 2 above tohave significant ATPase inhibition activity.

Two solutions were created as listed below. One contained theluciferase, luciferin, MgSO₄, and buffer (reagent composition). Theother solution contained media plus ATP (media solution).

Reagent Composition:

-   -   50 μg/ml enzyme (LucPpy is Promega catalog #E170A)    -   50 mM Hepes buffer (pH 7.0)    -   100 mM NaCl    -   1.0 mM EDTA    -   0.1% gelatin    -   10 mM MgSO₄    -   1.0 mM Luciferin    -   Detergent (at various concentrations, see table below)    -   Nanopure water was added to a final volume of 2.5 ml        Media Solution:    -   F12/DME media (Sigma D-6905) final volume 15 ml    -   1.0 μM ATP

For each reagent composition, all ingredients except the luciferaseenzyme were assembled; the enzyme was then added immediately before thefirst luminescence reading was taken. Immediately after addition ofenzyme to each reagent composition and for various timepointsthereafter, 100 μl aliquots of the reagent composition were added towells of a 96-well microtitre luminometer plate in triplicate. To thesewere added 100 μl Media Solution containing the ATP. Luminescence wasthen immediately read on a Dynex MLX microtitre plate luminometer, 0.5second reads per well. The average relative light unit values as well asthe reaction half lives are listed below in Table I and the percent oforiginal luminescence remaining at each timepoint is listed in Table Jbelow. The timepoint at the top of the column indicates the time atwhich the first RLU measurement for the samples in that column was read.Times are provided for some samples when their time of measurementvaried significantly from the time at the top of the column. Timeslisted in Table I are the same for Table J. The values for sulfobetaineand for CTAB were generated on different days, they are presented withthe control values generated in the same experiment. There wassubstantial precipitation in the samples containing 0.5% deoxycholateand slight precipitation in the samples containing 0.1% deoxycholate.This resulted in unreliable measurements for those samples. TABLE IAverage Relative Light Units LucPpv (at RT, pH 7.0) 0.75 min 28 min 39min 64 min 92 min Control (no det) 3620.263 3610.837 3530.333 3421.7433442.863 DTAB 0.5% 0.018 BDDABr 0.5% 0.001 *Deoxych. 0.5% 4.801 0.0120.007 SDS 0.05% 0.007 0.75 min 7.5 min 20.5 min 59 min 90 min Control(no det) 3238.01 3147.54 3192.85 3155.05 3203.21 Sulfobet. 1.0% 0.0240.000 Sulfobet. 0.5% 737.788 0.084 0.037 Sulfobet. 0.1% 3291.41 3178.073262.17 3074.49 3066.29 0.75 min 10 min 36 min 60 min 95 min Control (nodet) 2311.293 — 2294.607 2254.490 2196.130 DTAB 0.1% 0.307 0.009 0.005BDDABr 0.1% 0.007 SDS 0.01% 0.175 0.016 *Deoxych. 0.1% 19.771 0.1800.026 0.75 min 6 min 20 min 40 min 62 min 88 min Control (no det)2844.21 — 2672.94 2817.67 2869.35 2852.75 *CTAB 0.1% 94.603 0.020 *CTAB0.05% 1134.53 0.563 0.015 *CTAB 0.02% 1869.80 79.719 0.099 (16 min) 0.75min 38 min 70 min 94 min 122 min Control (no det) 3379.970 3194.5403085.880 3015.030 2820.330 DTAB 0.02% 4513.320 3761.940 3363.4103229.340 3082.030 BDDABr 0.02% 2894.700 0.035 0.015 0.016 0.011 Deoxych0.02% 3199.250 2844.010 2626.260 2496.570 2314.910 SDS 0.01% 17.5840.001 SDS 0.002% 640.290 190.860 84.210 47.210 25.630 LucPpe2m146 (atRT, pH 7.0) 15 min 72 min 124 min 190 min 251 min Control (no det)27668.0 25171.53 24023.967 23200.600 20771.450 DTAB 0.5% 5.496 3.1412.769 2.563 2.766 BDDABr 0.5% 2.040 1.140 0.998 0.899 1.042 *Deoxych.0.5% 45.977 229.874 2059.397 1414.397 1342.963 SDS 0.05% 0.018 0.0120.001 0.006 0.016 0.75 min 30 min 66 min 123 min 180 min 238 min Control(no det) 16897.5 16754.97 17594.7 16842.8 16935.5 17598.6 Sulfobet. 1.0%16897.5 16754.97 7036.0 6653.67 7358.44 6948.05 Sulfobet. 0.5% 13447.013597.97 14186.93 12982.20 14348.90 13958.2 Sulfobet. 0.1% 16648.317003.0 17849.8 16610.13 17115.63 18131.37 1.0 min 34 min 65 min 115 min175 min 238 min 286 min Control (no det) 27811.33 28665.87 28814.4726614.43 27752.97 28249.6 27110.13 DTAB 0.1% 20132.47 20574.63 20800.8019908.30 20760.93 19758.4 19659.10 BDDABr 0.1% 29.71 66.09 62.19 45.1833.89 34.46 126.90 *Deoxych. 0.1% 5150.43 5031.12 6855.67 6340.457347.09 6801.23 7472.54 SDS 0.01% 24.46 27.85 10.77 6.45 2.02 2.44 0.621.0 min 37 min 67 min 116 min 177 min 243 min Control (no det) 16578.615270.1 15710.6 16272.3 17085.8 17163.7 *CTAB 0.1% 1047.8 5.3 5.2 6.26.0 5.7 *CTAB 0.05% 11992.5 301.5 131.7 4219.7 16910.8 17340.0 *CTAB0.02% 11019.8 2051.7 1087.4 6117.8 16020.8 16368.5 1.0 min 34 min 65 min115 min 175 min 238 min 286 min Control (no det) 27811.33 28665.8728814.47 26614.43 27752.97 28249.6 27110.1 DTAB 0.02% 28730.17 28596.3028651.97 26062.30 27941.73 27677.9 27808.3 BDDABr 0.02% 26548.6026272.87 25766.43 24155.00 25891.70 24959.1 24510.1 Deoxych. 0.02%24307.30 24639.00 24717.97 23158.37 24655.43 24320.6 24303.9 SDS 0.002%24265.83 24213.97 24725.73 22654.87 23856.73 23425.5 23275.6*ppt indicates that the detergent precipitated in the sample

For each concentration of detergent using either luciferase, thestability of the reagent composition can be described as having anactivity half-life using the data from Table J. The half-life isdetermined by applying linear regression to the data, with the logarithmof the relative luminescence values in Table J as the dependentvariable, and the time of each measurement as the independent variable.The half-life is then calculated from the linear regression asln(0.5)/(slope). Using this method, the stabilities of the reagentcomposition are listed in Table K; detergent concentration shown aspercent (w/v) and values of activity half-life shown in minutes. Wherethe luminescence activity was less than could be reliably measured, thehalf-life is shown as “no activity.” Some reagent compositions were veryunstable, having activity half-lives less than 10 minutes. Because theseare difficult to accurately quantitate, they are listed only as “<10.”In contrast, some reagent compositions exhibited large stability, havingactivity half-lives greater than 1000 minutes (i.e., greater than 16hours). Because the samples were measured for fewer than 5 hours,accurate determinations of half-lives are difficult and are listed onlyas “>1000.” TABLE J Percent of Luminescence Remaining LucPpv (at RT, pH7.0) Control (no det) 100.00 99.74 97.52 94.52 95.10 DTAB 0.5% 100.00BDDABr 0.5% 100.00 Deoxych. 0.5% 100.00 0.25 0.15 (ppt) SDS 0.05% 100.00Control (no det) 100.00 97.2 98.6 108.5 97.4 98.9 Sulfobet. 0.5% 100.00Sulfobet. 0.1% 100.00 95.8 99.1 93.4 93.2 Control (no det) 100.00 nd99.28 97.54 95.02 DTAB 0.1% 100.00 2.93 1.55 BDDABr 0.1% 100.00 SDS0.01% 100.00 9.14 Deoxychol 0.1% 100.00 0.91 0.13 Control (no det)100.00 — 94.0 99.1 100.9 CTAB 0.1% 100.00 0.02 Ppt. CTAB 0.05% 100.000.05 0.00 Ppt. CTAB 0.02% 100.00 0.09 0.03 Ppt. Control (no det) 100.0094.51 91.30 89.20 83.44 DTAB 0.02% 100.00 99.53 74.52 71.55 68.29 BDDABr0.02% 100.00 0.00 0.00 Deoxych. 0.02% 100.00 88.90 82.09 78.04 72.36 SDS0.002% 100.00 29.81 13.15 7.37 4.00 LucPpe2m146 (at RT, pH 7.0) Control(no det) 100.00 90.98 86.83 83.85 75.07 DTAB 0.5% 100.00 57.16 50.3946.64 50.32 BDDABr 0.5% 100.00 55.90 48.93 44.07 51.10 Deoxychol 0.5%100.00 499.98 4479.22 3076.34 2920.97 Substantial precipitation SDS0.05% 100.00 65.68 6.09 35.06 87.64 Control (no det) 100.0 99.2 104.199.7 100.2 104.1 Sulfobet. 1.0% 100.00 102.0 106.0 100.3 110.9 104.7Sulfobet. 0.5% 100.00 101.1 105.5 96.5 106.7 103.8 Sulfobet. 0.1% 100.00102.1 107.2 99.8 102.8 108.9 Control (no det) 100.00 103.07 103.61 95.7099.79 101.58 97.48 DTAB 0.1% 100.00 102.20 103.32 98.89 103.12 98.1497.65 BDDABr 0.1% 100.00 222.47 209.32 152.09 114.06 115.98 427.13Deoxychol 0.1% 100.00 97.68 133.11 123.11 142.65 132.05 145.09 Precip.SDS 0.01% 100.00 113.85 44.02 26.38 8.25 9.97 2.53 Control (no det)100.00 92.11 94.76 98.15 103.06 103.53 CTAB 0.1% 100.00 0.51 0.50 0.590.58 0.55 Ppt CTAB 0.05% 100.00 2.51 1.10 35.19 141.01 144.59 Ppt CTAB0.02% 100.00 18.62 9.87 55.52 145.38 148.54 Ppt Control (no det) 100.00103.07 103.61 95.70 99.79 101.58 97.48 DTAB 0.02% 100.00 99.53 99.7390.71 97.26 96.34 96.79 BDDABr 0.02% 100.00 98.96 97.05 90.98 97.5394.01 92.32 Deoxych. 0.02% 100.00 101.36 101.69 95.27 101.43 100.0599.99 SDS 0.002% 100.00 99.79 101.90 93.36 98.31 96.54 95.92

In Table K, the values below the horizontal line (in bold) indicate theconcentrations of each detergent that inhibited endogenous ATPaseactivity in Examples 1 and 2 by at least 25%. It is clear that thereagent composition is very unstable when comprising LucPpy and has aconcentration of detergent capable of inhibiting at least 25% of theendogenous ATPase activity. In contrast, a reagent comprising LucPpe2m146 and the same detergent concentration generally has moderate tosubstantial stability. In some cases, luciferase activity was inhibitedby the presence of the detergent, but nonetheless yielded a stablecomposition. Two detergents, CTAB and deoxycholate, precipitated fromthe solution during the course of the measurements. This was mostnotable for CTAB, where LucPpe2 m146 was strongly inactivated by thedetergent, but slowly regained activity as the detergent precipitatedfrom solution. This behavior made half-life impossible to estimate forthe reagent composition containing CTAB. The effect was seen to a lesserextent with deoxycholate. TABLE K Half-life of Luminescence Activity(measured in minutes) conc. DTAB CTAB BDDABr Deoxych. SDS SulfobetaineCommon firefly luciferase (P. pyralis)0.000 >1000 >1000 >1000 >1000 >1000 >1000 0.002         25 0.010                      <10 0.020       193       <10*  <10  235 0.050 <10*            no activity 0.100 <10 <10* no activity <10*  877 0.2000.500 no activity no activity <10*       no activity 1.000 Chemostableluciferase (Ppe2m146) 0.000 >1000 >1000 >1000 >1000 >1000 0.002 >10000.010                         83 0.020 >1000 * >1000 >1000 0.050 *           0.100 >1000 * >1000 >1000* >1000 0.200 0.500  265  264>1000* >1000         1.000 >1000*precipitate formed

Example 4 ATPase Inhibition at 22° C. and 37° C.

Cell lysate was prepared as described in Example 1. The ability of 1%DTAB to inhibit endogenous ATPase (i.e., ATPase present in a sample) wasmeasured in a complete reagent composition over time at both 22° C.(room temperature) and 37° C. Three cell lysate samples were prepared.Sample 1 contained 4.0 ml L929 cell lysate plus 4.0 ml 25 mM Hepes, pH7.5. Sample 2 contained 4.0 ml L929 cell lysate plus 4.0 ml buffereddetergent solution (40 mM Citrate (pH 6.0), 110 mM MES (pH 6.0), 450 mMKPO4 (pH 6.0), 2.0 mM EDTA, 0.2% Mazu DF-204 (PPG Industries), 2.0 mMNaF, 1.0% DTAB and 2% THESIT®—final pH of the buffered detergentsolution adjusted to 6.0). Sample 3 contained 4.0 ml DMEM/F12 media (noserum) containing 0.1 μM ATP plus 4.0 ml 25 mM Hepes (pH 7.5). Thesamples were divided in half—half was incubated at 22° C.; half wasincubated at 37° C. After 10 minutes of incubation in their respectivewater baths to allow the solution at 37° C. to reach that temperature(this point is referred to as time=10 min.), 100 μl of each sample wastransferred independently to a well of a 96-well luminometer plate inquadruplicate. To each 100 μl sample was added 20 μl of a solutioncontaining luciferin (12.5 mM)/luciferase (200 μg/ml LucPpe2 m146)/MgSO₄(50 mM) solution. The resulting relative light units were measured on aDynex luminometer using 0.5 second reads. This was repeated at severaltime points out to five hours. The resulting average relative light unitvalues (Table L) and the percent remaining ATP (Table M) are below.TABLE L Average Relative Light Unit Values Time (minutes) Temp. 10 min60 min 110 min 180 min 240 min 300 min 37° C. Sample 1 12371.2 3141.2930.0 270.7 127.4 55.1 Sample 2 781.3 792.3 777.5 748.9 728.2 713.4Sample 3 2937.2 2815.0 2869.2 2592.3 2046 1953.4 22° C. Sample 1 13073.45551.8 2766.29 1121.5 541.8 273.7 Sample 2 606.6 615.5 630.0 615.7 616.4626.0 Sample 3 2603.4 2527.0 2533.0 2321.9 2052.8 1917.3

TABLE M Percent ATP Remaining Relative light units Temp 10 min 60 min110 min 180 min 240 min 300 min 37° C. Sample 1 100 25.39 7.52 2.19 1.030.44 Sample 2 100 101.41 99.51 95.86 93.20 91.31 Sample 3 100 95.8497.69 88.26 69.68 66.51 22° C. Sample 1 100 42.47 21.16 8.58 4.14 2.09Sample 2 100 101.46 103.86 101.5 101.62 103.21 Sample 3 100 97.07 97.3089.19 78.85 73.65Under the conditions of this assay, 1% DTAB resulted in no loss of ATP(i.e., complete inhibition of ATPase endogenous to the sample) at 22° C.and minimal loss at 37° C., even when the solution was incubated at thetemperature of interest for up to five hours. These data demonstratereaction conditions for which there is nearly complete endogenous ATPaseinhibition, yet in which ATP is stable to at least five hours.

Example 5 III. Stability of Reagent Composition

Using the results of Example 4, this experiment was designed todemonstrate the stability of the complete reagent composition at 22° C.in comparison to its stability at 37° C. as measured by luminescenceover time. To generate the complete reagent composition, 10 ml ofbuffered detergent (36 mM Sodium Citrate, 2 mM EDTA, 20 mM MgSO4, 2 mMNaF, 1% DTAB, 2% THESIT®, 0.2% Mazu, buffered to a final pH of 6.0) wasadded to lyophilized LucPpe2 m146, D-luciferin, MgSO4, CDTA so that thereagent composition had a final luciferase concentration of 80 μg/ml andfinal luciferin concentration of 5 mM.

The reagent composition was divided in half with one half incubated at22° C. and the other half incubated in a water bath at 37° C. At varioustimepoints, 100 μl samples were removed from the reagent compositions(in quadruplicate) and transferred to a 96 well luminometer plate. Then,to each 100 μl sample was added 100 μl of 1.0 μM ATP in DPBS (Dulbecco'sPhosphate Buffered Saline, Sigma Corp., St. Louis, Mo.). The plate wasshaken for 30 seconds at 700 rpm on an orbital shaker and luminescencewas then read on a Dynex MLX microtiter plate luminometer, 0.5 secondreads per well. The average relative light unit values are listed belowin Table N and the percent remaining ATP and half life values in Table0. TABLE N Average Relative Light Units Time (min.): 0 23 60 120 185 240300 22° C. 695.4 763.0 741.8 683.8 675.9 664.4 681.4 37° C. — 757.7722.3 614.4 575.0 544.2 526.1

TABLE O Percent Remaining ATP Time (min.): 0 23 60 120 185 240 300half-life 22° C. 100 109.7 106.7 98.3 97.2 95.5 98.0 >1000 min 37° C.100 108.9 103.9 88.4 82.7 78.2 75.7 573 min

Example 6 DTAB Effect on Stability of Reagent Compositions ComprisingVarious Luciferases (37° C.)

The stability of a reagent composition comprising LucPpe2 luciferase wascompared to a reagent composition comprising LucPpe2 m90 and a reagentcomposition comprising LucPpe2 m146, in the presence and absence of 0.1%DTAB. These mutant luciferases are thermostable; they are described indetail in PCT application PCT/US99/30925, filed Dec. 22, 1999.

Each enzyme was diluted to 0.05 mg/ml to a final volume of 1.0 ml ineither 25 mM HEPES, pH 8.0 or 20 mM citrate, pH 6.0; both with 100 mMNaCl, 1 mM EDTA, 0.1% gelatin and 5% glycerol. In half of the samples,0.1% DTAB was added. The enzyme solutions (“reagent compositions”) wereincubated at 37° C.

At various timepoints, 10 μl of enzyme solution was transferred to a96-well luminometer plate in triplicate. Then, in triplicate, 100 μlroom temperature luciferase assay reagent (1 mM luciferin, 0.2 mM ATP,10 mM MgSO₄ in 50 mM HEPES, pH 8.0) was added to each 10 μl enzymesolution aliquot, mixed and immediately read in a Dynex MLX microtiterplate luminometer. The ATP in the luciferase assay reagent is at asaturated concentration. The average RLU values are reported, as are thehalf lives of the reagent composition activity as measured byluminescence (Table P). Half-life was calculated using the formula log(0.5)/slope of the data plotted as time (x axis) versus log value ofRLU. The time 0 is actually about 2-3 minutes after the mixing of enzymeand substrate; this could account for the lower numbers in the presenceof DTAB at time 0 than in the absence of DTAB. TABLE P Effect of DTAB onvarious luciferases' activity Average RLU over time (min) Enzyme/time pH6.0 + pH 8.0 + (min) pH 6.0 DTAB pH 8.0 DTAB LucPpe2 0 18655.25 2372.3122134.0 7048.95 29 1854.83 0.099 11773.67 0.161 59 206.69 0.08 5070.620.034 Half life (min) 9.1 4.0 27.7 3.4 LucPpe2m90 0 20031.03 16263.5315795.17 22401.4 59 18453 15535.4 15510.77 15332.67 123 15966.03 13421.513735.67 12383.75 183 15546.4 14061.8 13271.05 11271.25 239 14519.6311924.03 11115.13 9551.65 Half life (min) 510.5 580.1 487.5 207.1LucPpe2m146 0 7685.01 5945.34 6652.84 6445.57 61 7077.29 5989.65 6577.916214.56 125 5507.01 3754.71 5192.73 4018.92 183 6144.94 4839.58 5476.864020.56 237 6471.76 4951.0 5174.22 3734.59 Half life (min) 820.8 682.6595.5 268.0*time 0 is about 2-3 minutes after mixing of enzyme and substrate

Example 7 Enhanced Duration of Luminescence and Effects of Various Mediaand Sera on ATP Measurement

In some embodiments, the compositions of the invention were added tointact cells to lyse them, and then ATP detected. In other embodiments,conditioned culture media were themselves assayed for ATP. However, thevarious components of media, such as buffers, sugars, amino acids, pHindicators, salts, etc., as well as the various factors found in serum(equine, bovine, etc.) may inhibit luciferase activity. This exampledemonstrates the effect of cell culture media and sera in the presenceof the ATPase inhibitor DTAB on the reagent composition and on theduration of luminescence (referred to herein as “signal stability”) whenthe reagent composition is combined with a sample supplying ATP.

The following reagent composition was prepared: 40 mM Citrate buffer (pH6.0), 110 mM MES buffer (pH 6.0), 0.2 mM EDTA, 100 μg/ml Luciferase(LucPpe2 m146, diluted from 37.8 mg/ml stock solution), 5 mM luciferin,300 mM NaCl, 20 mM MgSO₄, 0.05% Mazu DF-204, and varying concentrationsDTAB as listed in Table Q below. The luciferase-luciferin reaction wasinitiated by combining 100 μl of the reagent composition with 100 μlcell media with serum, varying final concentrations of DTAB (asindicated below in Table Q) and 1.0 μM ATP was added to each reactionplus or minus 10 μM sodium pyrophosphate in wells of a 96-wellmicrotiter luminometer plate; each experimental condition was preparedin triplicate. At various times after initiating the reaction, RLUvalues were recorded using an Orion microplate luminometer (BertholdDetection Systems; Pforzheim, Germany). Average RLUs and signalstability (measured in terms of its half-life) are reported in Table Q.In all media tested, the DTAB in the presence of Ppi resulted in alonger half-life than did the DTAB in the absence of the Ppi. The typeof media used in the assay did not contribute significantly to signalstability variation. The signal stability half life was calculated fromthe time 0 (A) and from time 10 min as the original value (B). The Ppidecreased luminescence about ten-fold or more. TABLE Q Effects of mediaand sera on luciferase-luciferin reaction Signal stability Averagerelative light units over time (min) t_(1/2) (hr) Conditions¹ 0 10 30 65120 180 325 A B DMEM + 10% FBS + ATP + 0.50% DTAB 204771 223493 187757146766 104150 72609 32307 1.9 1.9 0.55% DTAB 52107 59799 51285 4215532891 24137 12031 2.4 2.3 0.45% DTAB/Ppi 4989 4723 4053 3743 3611 35143188 9.6 11.4 F12/DME + 10% FBS + ATP + 0.50% DTAB 13467 13311 113489953 8127 6538 3793 3.0 3.0 0.55% DTAB 6847 6952 6013 5581 4891 43293207 4.9 5.0 0.45% DTAB/Ppi 1467 1342 1107 1047 1017 1036 937 10.6 14.2F12/DME + 10% HS + ATP + 0.50% DTAB 88405 82645 70746 58749 44677 3335317414 2.3 2.4 0.55% DTAB 36615 36669 31348 27979 23778 19955 13539 3.83.8 0.45% DTAB/Ppi 6124 5611 4981 4398 4163 3961 3553 7.7 9.0 RPMI + 10%FBS + ATP 0.50% DTAB 44179 43664 35909 26501 16951 10153 3268 1.4 1.40.55% DTAB 21388 22863 19631 16848 12968 9661 5080 2.5 2.5 0.45%DTAB/Ppi 3284 2879 2599 2381 2285 2243 2099 10.4 13.6

Example 8 Cell Number Correlates with Light Output

This experiment demonstrates that luminescence generated by use of thereagent composition in the method of the invention directly correlateswith viable cell number. A simple correlation between known living-cellnumbers and experimentally-determined luminescence was established.

Jurkat cells (ATCC, CRL-1990) were grown in 5% CO₂/95% air, 100%humidity at 37° C. and maintained in RPMI media (Sigma, R-8005)containing 10% FBS (Hyclone #SH30070), 1× non-essential amino acids(Hyclone SH30238) and 1 mM sodium pyruvate (Hyclone #SH30239). Cellswere suspended at 5×10⁵/ml in fresh complete medium, and 1:2 serialdilutions were prepared. Then, 100 μl of the cell dilutions were addedto wells of a 96-well microtiter plate, resulting in 0-50,000cells/well. Quadruplicate replicates were prepared. The plate was thenincubated at 37° C., 5% CO₂ for 45 minutes. The plate was thenequilibrated at 22° C. for 30 minutes. Then 100 μl reagent composition(40 mM Citrate buffer (pH 6.0), 110 mM MES buffer (pH 6.0), 2 mM EDTA,450 mM KPO₄, 0.4% Prionex, 80 μg/ml Luciferase (LucPpe2 m146, dilutedfrom 37.8 mg/ml stock solution), 5 mM luciferin, 2% THESIT®, 20 mM NaF,20 mM MgSO₄, 0.2% Mazu DF-204, 1.0% DTAB), was added to each well, theplate was gently shaken for 2 minutes, and incubated for 10 minutes on aDynex MLX plate luminometer. The light output was then read in 0.5second summed interrogations. The resulting average RLUs are reportedherein below in Table R. TABLE R Correlation of luminescence with cellnumber Luminescence standard Cells/well (RLU) deviation 0 0.07 0.015 491.96 0.233 98 3.27 0.460 195 6.71 0.307 390 12.34 0.356 781 23.53 0.3671562 47.12 1.583 3125 91.77 1.156 6250 171.93 0.812 12500 346.26 10.73925000 672.79 7.322 50000 1279.75 14.683

Example 9 Lymphoid Cells Tested with NaF

In some cells, such as lymphoid cells (e.g., Jurkat), an increase inluminescence over time in the presence of the reagent compositionsolution is observed. While the underlying mechanism of this increase inluminescence is not known, it is postulated that it results from thefunctioning of ATP-generating enzymes in this cell-type. The activity ofsuch enzymes, if left unchecked, will result in an overestimation of ATPin a sample at the time of the assay. This experiment was designed totest the effects of sodium fluoride on luminescence when using lymphoidcells. The experiment also demonstrates the enhanced duration ofluminescence produced by the LucPpe2 m146 in the composition and methodsof the invention.

Jurkat cells (ATCC, CRL-1990) were grown in 5% CO₂/95% air, 100%humidity at 37° C. and maintained in RPMI media (Sigma, R-8005)containing 10% FBS (Hyclone #SH30070), 1× non-essential amino acids(Hyclone SH30238) and 1 mM sodium pyruvate (Hyclone #SH30239). Cellswere plated at 0, 12500, 25000, and 50000 cells/well in 100 μl of mediain a 96-well microtiter luminometer plate. Quadruplicate replicates wereprepared. To each of these wells was added reagent composition (reagentcomposition=40 mM Citrate buffer (pH 6.0), 110 mM MES buffer (pH 6.0),0.2 mM EDTA, 0.2% Gelatin, 100 μg/ml Luciferase (LucPpe2 m146, dilutedfrom 37.8 mg/ml stock solution), 100 μM luciferin, 300 mM NaCl, 20 mMMgSO₄, 0.05% Mazu DF-204, 0.6% DTAB). In addition, all but the “no KPO₄”control sample contained 60 mM KPO₄ buffer (pH 6.0). Variousconcentrations of NaF were then added to the above solution to finalconcentrations of 0, 1.0, 2.0, 4.0, 10.0 mM; one condition had 10.0 mMNaF but without KPO₄.

Total reaction volume per well was 200 μl, consisting of 100 μl cellsplus media and 100 μl reagent composition containing KPO₄ and/or NaF.The light output was taken at various times on a Dynex microtiter plateluminometer at a 0.5 second read time. The resulting average RLUs fromquadruplicate wells and calculated signal stabilities as measured bytheir half-life values are reported in Table S.

The data demonstrate that the addition of NaF can inhibit theluminescence increase seen when using Jurkat cells in a method of thepresent invention. TABLE S Effect of NaF on Jurkat cell luminescenceAverage RLU over time (min) t_(1/2) Cells/Well 0 30 85 130 220 255 285(hrs) Control 0 2.84 2.77 3.28 4.30 3.98 4.00 3.97 — 12500 355.70 370.90420.87 470.88 508.06 523.80 536.11 nc* 25000 761.86 857.57 1174.301599.85 2709.75 3099.69 3276.71 nc 50000 1409.87 1946.82 5067.5412596.83 4991.96 3583.77 2978.22 nc 1.0 mM NaF (in the reagentcomposition) 0 2.87 2.72 2.76 2.91 2.42 2.37 2.27 — 12500 358.61 341.31331.08 323.54 271.89 257.06 247.61 8.8 25000 691.68 640.53 611.45 596.87495.89 463.32 446.32 7.6 50000 1380.16 1268.49 1185.73 1147.33 936.82862.31 832.15 6.6 2.0 mM NaF 0 2.80 2.59 2.58 2.57 2.07 2.06 1.95 —12500 348.16 333.48 323.75 317.48 258.43 251.90 239.40 8.6 25000 679.02631.69 603.07 589.08 472.37 455.25 433.54 7.3 50000 1340.22 1239.831156.03 1111.04 873.26 829.87 781.80 6.1 4.0 mM NaF 0 6.55 7.65 15.3033.49 15.12 11.87 10.43 — 12500 367.69 346.05 335.27 331.07 275.13259.58 251.79 8.6 25000 691.41 634.08 604.37 587.41 476.36 453.66 436.387.2 50000 1378.13 1277.41 1195.56 1139.99 883.49 853.43 813.31 6.1 10.0mM NaF 0 6.55 6.20 6.47 7.87 5.52 5.27 5.13 — 12500 364.79 343.44 331.48326.32 271.64 256.88 248.30 8.5 25000 753.29 677.94 635.67 617.77 496.08471.66 450.76 6.6 50000 1442.08 1324.67 1235.80 1181.84 903.44 870.95826.48 5.8 10.0 mM NaF, without KPO₄ 0 5.95 5.65 5.43 5.34 4.19 4.163.91 — 12500 409.57 400.10 384.84 371.42 294.12 283.01 267.78 7.3 25000806.87 785.16 734.44 692.57 516.69 498.99 468.90 5.7 50000 1514.801449.30 1340.91 1258.88 889.88 865.24 807.41 4.9*nc - not calculable due to increase in RLU values over time

REFERENCES

-   Aiginger, P., R. Kuzmits, H. Lang, and M. M. Muller. 1980. Changes    in the ATP content of leukaemic cells induced by cytotoxic    substances. J. Clin. Chem. Clin. Biocehm. 1:216.-   Andreotti, P. E., I. A. Cree, C. M. Kurbacher, D. M. Hartmann, D.    Linder, G. Harel, I. Gleiberman, P. A. Caruso, S. H. Ricks, M.    Untch, and et al. 1995. Chemosensitivity testing of human tumors    using a microplate adenosine triphosphate luminescence assay:    clinical correlation for cisplatin resistance of ovarian carcinoma.    Cancer Res. 55:5276-82.-   Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.    Seidman, J. A. Smith, and K. Struhl. 1987. Current protocols in    molecular biology. John Wiley & Sons, New York.-   Baldwin, T. O., and V. A. Green. 2000. Purification of firefly    luciferase from recombinant sources. Methods Enzymol. 305:180-8.-   Beny, M., and M. Dolivo. 1976. Separation of firefly luciferase    using an anion exchanger. FEBS Lett. 70:167-70.-   Bostick, W. D., M. S. Denton, and S. R. Dinsmore. 1982.    Liquid-chromatographic separation and bioluminescent detection of    creatine kinase isoenzymes. In Bioluminescence and    Chemiluminescence: Instruments and Applications. Vol. II. K. Van    Dyke, editor. CRC Press, Boca Raton, Fla. 227-246.-   Bowie, L. J., V. Horak, and M. De Luca. 1973. Synthesis of a new    substrate analog of firefly luciferin. An active-site probe.    Biochemistry. 12:1845-52.-   Bradbury, D. A., T. D. Simmons, K. J. Slater, and S. P.    Crouch. 2000. Measurement of the ADP:ATP ratio in human leukaemic    cell lines can be used as an indicator of cell viability, necrosis    and apoptosis. J Immunol Methods. 240:79-92.-   Branchini, B. R. 2000. Chemical synthesis of firefly luciferin    analogs and inhibitors. Methods Enzymol. 305:188-95.-   Branchini, B. R., M. M. Hayward, S. Bamford, P. M. Brennan,    and E. J. Lajiness. 1989. Naphthyl- and quinolylluciferin: green and    red light emitting firefly luciferin analogues. Photochem Photobiol.    49:689-95.-   Branchini, B. R., T. M. Marschner, and A. M. Montemurro. 1980. A    convenient affinity chromatography-based purification of firefly    luciferase. Anal Biochem. 104:386-96.-   Carter, P. 1986. Site-directed mutagenesis. Biochem J. 237:1-7.-   Craig, F. F., A. C. Simmonds, D. Watmore, F. McCapra, and M. R.    White. 1991. Membrane-permeable luciferin esters for assay of    firefly luciferase in live intact cells. Biochem J. 276:637-41.-   Cree, I. A. 1998. Luminescence-based cell viability testing. Methods    Mol. Biol. 102:169-77.-   Cree, I. A., and P. E. Andreotti. 1997. Measurement of Cytotoxicity    by ATP-based Luminescence Assay in Primary Cell Cultures and Cell    Lines. Toxicology in Vitro. 11:553-556.-   Crouch, S. P., R. Kozlowski, K. J. Slater, and J. Fletcher. 1993.    The use of ATP bioluminescence as a measure of cell proliferation    and cytotoxicity. J Immunol Methods. 160:81-8.-   Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 1966. A    proposal for a uniform nomenclature in bacterial genetics. Genetics.    54:61-76.-   Ebadi, M. S. 1972. Firefly luminescence in the assay of cyclic AMP.    Adv Cyclic Nucleotide Res. 2:89-109.-   Filippova, N. Y., A. F. Dukhovich, and N. N. Ugarova. 1989. New    approaches to the preparation and application of firefly luciferase.    J Biolumin Chemilumin. 4:419-22.-   Hastings, J. W. 1996. Chemistries and colors of bioluminescent    reactions: a review. Gene. 173:5-11.-   Hastings, J. W., and T. Wilson. 1976. Bioluminescence and    chemiluminescence. Photochem Photobiol. 23:461-73.-   Jassim, S. A., A. Ellison, S. P. Denyer, and G. S. Stewart. 1990. In    vivo bioluminescence: a cellular reporter for research and industry.    J Biolumin Chemilumin. 5:115-22.-   Jones, K., F. Hibbert, and M. Keenan. 1999. Glowing jellyfish,    luminescence and a molecule called coelenterazine. Trends    Biotechnol. 17:477-81.-   Kajiyama, N., and E. Nakano. 1993. Thermostabilization of firefly    luciferase by a single amino acid substitution at position 217.    Biochemistry. 32:13795-9.-   Kajiyama, N., and E. Nakano. 1994. Enhancement of thermostability of    firefly luciferase from Luciola lateralis by a single amino acid    substitution. Biosci Biotechnol Biochem. 58:1170-1.-   Kangas, L., M. Gronroos, and A. L. Nieminen. 1984. Bioluminescence    of cellular ATP: a new method for evaluating cytotoxic agents in    vitro. Med Biol. 62:338-43.-   Kiechle, F. L., L. Jarett, D. A. Popp, and N. Kotagal. 1980.    Isolation from rat adipocytes of a chemical mediator for insulin    activation of pyruvate dehydrogenase. Diabetes. 29:852-5.-   Kricka, L. J., and M. De Luca. 1982. Effect of solvents on the    catalytic activity of firefly luciferase. Arch Biochem Biophys.    217:674-81.-   Lundin, A., J. Anson, and P. Kau. 1994. ATP extractants neutralised    by cyclodextrins. In Bioluminescence and Chemiluminescence:    Fundamental and Applied Aspects. A. K. Campbell, L. J. Kricka,    and P. E. Stanley, editors. John Wily & Sons, New York. 399-402.-   McElroy, W. D., H. H. Seliger, and E. H. White. 1969. Mechanism of    bioluminescence, chemiluminescence and enzyme function in the    oxidation of firefly luciferin. Photochem Photobiol. 10:153-70.-   Miska, W., and R. Geiger. 1987. Synthesis and characterization of    luciferin derivatives for use in bioluminescence enhanced enzyme    immunoassays. New ultrasensitive detection systems for enzyme    immunoassays, I. J Clin Chem Clin Biochem. 25:23-30.-   Missiaen, L., F. Wuytack, H. De Smedt, M. Vrolix, and R.    Casteels. 1988. AlF4-reversibly inhibits ‘P’-type cation-transport    ATPases, possibly by interacting with the phosphate-binding site of    the ATPase. Biochem J. 253:827-33.-   Morii, M., and N. Takeguchi. 1993. Different biochemical modes of    action of two irreversible H+, K(+)-ATPase inhibitors, omeprazole    and E3810. J Biol Chem. 268:21553-9.-   Moyer, J. D., and J. F. Henderson. 1983. Nucleoside triphosphate    specificity of firefly luciferase. Anal Biochem. 131:187-9.-   Nguyen, V. T., M. Morange, and O. Bensaude. 1988. Firefly luciferase    luminescence assays using scintillation counters for quantitation in    transfected mammalian cells. Anal Biochem. 171:404-8.-   Petty, R. D., L. A. Sutherland, E. M. Hunter, and I. A. Cree. 1995.    Comparison of MTT and ATP-based assays for the measurement of viable    cell number. J Biolumin Chemilumin. 10:29-34.-   Picciolo, G. L., E. W. Chappelle, R. R. Thomas, and M. A.    McGarry. 1977. Performance characteristics of a new photometer with    a moving filter tape for luminescence assay. Appl Environ Microbiol.    34:720-4.-   Ronner, P., E. Friel, K. Czemiawski, and S. Frankle. 1999.    Luminometric assays of ATP, phosphocreatine, and creatine for    estimation of free ADP and free AMP. Anal Biochem. 275:208-16.-   Sambrook, J. 1989. Molecular cloning: a laboratory manual. Cold    Spring Harbor Laboratory, Cold Spring Harbor.-   U.S. Pat. No. 5,618,682.1997. BIOLUMINESCENCE MEASUREMENT SYSTEM.    US.-   U.S. Pat. No. 5,866,348.1999. BIOLUMINESCENCE MEASUREMENT SYSTEM.    US.-   Simpson, W. J., and J. R. Hammond. 1991. The effect of detergents on    firefly luciferase reactions [published erratum appears in J    Biolumin Chemilumin 1991 July-September;6(3):146]. J Biolumin    Chemilumin. 6:97-106.-   Stanley, P. E. 1989. A review of bioluminescent ATP techniques in    rapid microbiology. J Biolumin Chemilumin. 4:375-80.-   Thomson, C. M., P. J. Herring, and A. K. Campbell. 1997. The    widespread occurrence and tissue distribution of the    imidazolopyrazine luciferins. J Biolumin Chemilumin. 12:87-91.-   WO 00/18953. 2000. Method for detecting ATP. PCT.-   Vaskinn, S., E. Sundkvist, R. Jaeger, and G. Sager. 1999. The effect    of Mg2+, nucleotides and ATPase inhibitors on the uptake of    [3H]-cGMP to inside-out vesicles from human erythrocytes. Mol Membr    Biol. 16:181-8.-   Wells, J. A., M. Vasser, and D. B. Powers. 1985. Cassette    mutagenesis: an efficient method for generation of multiple    mutations at defined sites. Gene. 34:315-23.-   White, E. H., E. Rapaport, T. A. Hopkins, and H. H. Seliger. 1969.    Chemi- and bioluminescence of firefly luciferin. J Am Chem Soc.    91:2178-80.-   White, H. E., J. D. Miano, and M. Umbreit. 1975. Letter: on the    mechanism of firefly luciferin luminescence. J Am Chem Soc.    97:198-200.-   White, P. J., D. J. Squirrell, P. Arnaud, C. R. Lowe, and J. A.    Murray. 1996. Improved thermostability of the North American firefly    luciferase: saturation mutagenesis at position 354. Biochem J.    319:343-50.-   Wilson, T., and J. W. Hastings. 1998. Bioluminescence. Annu Rev Cell    Dev Biol. 14:197-230.-   U.S. Pat. No. 5,283,179.1994. LUCIFERASE ASSAY METHOD. US.-   U.S. Pat. No. 5,650,289.1997. LUCIFERASE ASSAY COMPOSITIONS. US.-   WO 9914336. 1999. Thermostable luciferases and methods of    production. PCT.-   Wood, K. V., Y. A. Lam, and W. D. McElroy. 1989. Introduction to    beetle luciferases and their applications. J Biolumin Chemilumin.    4:289-301.-   Yang, J., and D. B. Thomason. 1993. An easily synthesized,    photolyzable luciferase substrate for in vivo luciferase activity    measurement. Biotechniques. 15:848-50.-   Ye, L., L. M. Buck, H. J. Schaeffer, and F. R. Leach. 1997. Cloning    and sequencing of a cDNA for firefly luciferase from photuris    pennsylvanica. Biochim Biophys Acta. 1339:39-52.-   Zoller, M. J., and M. Smith. 1987. Oligonucleotide-directed    mutagenesis: a simple method using two oligonucleotide primers and a    single-stranded DNA template. Methods Enzymol. 154:329-50.

1. A kit for detecting ATP comprising: a luciferase component comprisinga chemostable luciferase separated from an ATPase inhibitor componentcomprising one or more ATPase inhibitors, where the combination of theluciferase component and the ATPase inhibitor component prior tocombination with a sample containing ATP forms a reagent composition fordetecting the ATP in the sample; where the reagent composition maintainsat least about 30% activity, as measured by luminescence after thereagent composition is combined with the sample, for at least one hourafter the reagent composition is combined with the sample containingATP, as compared to the reagent composition's activity just after theluciferase is combined with the ATPase inhibitor; and where the ATPaseinhibitor component present in the reagent composition is able to reduceATPase activity endogenous to the sample by at least about 25% relativeto the sample's ATPase activity in the absence of the ATPase inhibitorcomponent.
 2. The kit of claim 1, where the luciferase componentcomprises an anhydrous preparation of a chemostable luciferase.
 3. Thekit of claim 2, where the luciferase component comprises a lipholizedchemostable luciferase.
 4. The kit of claim 1, where the luciferasecomponent comprises an aqueous preparation of a chemostable luciferase5. The kit of claim 1, where the luciferase comprises an amino acidsequence selected from the group consisting of SEQ ID NOs.: 1, 2, 3, and4
 6. The kit of claim 1, where the kit further comprises a luciferinthat is separated from the ATPase inhibitor component.
 7. The kit ofclaim 6, where the luciferase component comprises the luciferin.
 8. Thekit of claim 1, where at least one ATPase inhibitor comprises adetergent.
 9. The kit of claim 8, where at least one detergent is acationic detergent.
 10. The kit of claim 9, wherein the cationicdetergent is selected from the group consisting ofdodecyltrimethylammonium bromide and benzyldimethyldodecylammoniumbromide.
 11. The kit of claim 8, where at least one detergent is ananionic detergent.
 12. The kit of claim 11, where the anionic detergentis selected from the group consisting of: SDS and deoxycholate.
 13. Thekit of claim 8, where at least one detergent is a zwitterionicdetergent.
 14. The kit of claim 13, where the zwitterionic detergent issulfobetaine 3-10.
 15. The kit of claim 1, where the kit furthercomprises an enzyme stabilizing agent.
 16. The kit of claim 15, wherethe enzyme stabilizing agent is selected from the group consisting of:2% polyethylene glycol 400 dodecyl ether, bovine serum, albumin, andgelatin.
 17. The kit of claim 16, where the enzyme stabilizing agent isseparated from the luciferase component.
 18. The kit of claim 1, wherethe kit further comprises a cell lysing agent separated from theluciferase component.
 19. The kit of claim 18, where the ATPaseinhibitor component comprises the cell lysing agent.
 20. The kit ofclaim 1, where the kit further comprises an ATP extracting agentseparated from the luciferase component.
 21. The kit of claim 20, wherethe ATPase inhibitor component comprises the ATP extracting agent. 22.The kit of claim 20, where the ATP extracting agent comprises CTAB. 23.The kit of claim 1, wherein the kit further comprises a luminescenceduration enhancing substance.
 24. The kit of claim 23, where the ATPaseinhibitor component comprises the luminescence duration enhancingsubstance, and the luminescence duration enhancing substance is selectedfrom the group consisting of: co-enzyme A, dithiothreitol,β-mercaptoethanol, EDTA, protease inhibitors and high saltconcentrations.
 25. The kit of claim 1, where the kit further comprisesan inhibitor of ATP-generating enzymes, the inhibitor separated from theluciferase component.
 26. The kit of claim 25, where the inhibitor ofATP-generating enzymes is selected from the group consisting of: NaF,vanadate, paranitrophenyl phosphate and dichloroacetic acid.
 27. The kitof claim 25, where the inhibitor of ATP-generating enzymes is NaF. 28.The kit of claim 25, where the ATPase inhibitor component comprises theinhibitor of ATP generating enzymes.
 29. The kit of claim 1, where theATPase inhibitor component further comprises a buffer, a divalent cationmetal chelator and magnesium cations.
 30. The kit of claim 1, where thekit further comprises instructions to admix the luciferase component andthe ATPase inhibitor component.
 31. The kit of claim 30, where theinstructions further provide the step of contacting a sample with areagent comprising a mixture of the luciferase component and the ATPaseinhibitor component.
 32. The kit of claim 30, where the instructions areprovided in a form selected from the group consisting of: instructionsprinted on the packaging, instructions printed in an instruction bookletor insert within the packaging, instructions in an electronic-readablemedium, and instructions accessable via the internet.
 33. A kit fordetecting ATP comprising: a luciferase component comprising a luciferaseseparately stored from ATPase inhibitor component comprising one or moreATPase inhibitors, where the combination of the luciferase component andthe ATPase inhibitor component forms a reagent composition for detectingATP in a sample; where the reagent composition maintains at least about30% activity, as measured by luminescence after the reagent compositionis combined with the sample, for at least one hour after the reagentcomposition is combined with a sample containing ATP, as compared to thereagent composition's activity just after the luciferase is combinedwith the one or more ATPase inhibitors, and where the one or more ATPaseinhibitors present in the reagent composition are collectively able toreduce ATPase activity endogenous to the sample by at least about 25%relative to the sample's ATPase activity in the absence of the one ormore ATPase inhibitors.
 34. The kit of claim 33 wherein the where theATPase inhibitor comprises a cationic detergent that is present in thereagent composition at a concentration of at least 0.1% (w/v).
 35. Thekit of claim 33 where the reagent composition maintains at least about60% activity, as measured by luminescence after the reagent compositionis combined with the sample, for at least one hour after the reagentcomposition is combined with a sample containing ATP, as compared to thereagent composition's activity just after the luciferase is combinedwith the one or more ATPase inhibitors, and where the one or more ATPaseinhibitors present in the reagent composition are collectively able toreduce ATPase activity endogenous to the sample by at least about 25%relative to the sample's ATPase activity in the absence of the one ormore ATPase inhibitors.
 36. The kit of claim 33, where the reagentcomposition maintains at least about 30% activity, as measured byluminescence after the reagent composition is combined with the sample,for at least one hour after the reagent composition is combined with asample containing ATP, as compared to the reagent composition's activityjust after the luciferase is combined with the one or more ATPaseinhibitors, and where the one or more ATPase inhibitors present in thereagent composition are collectively able to reduce ATPase activityendogenous to the sample by at least about 40% relative to the sample'sATPase activity in the absence of the one or more ATPase inhibitors. 37.The kit of claim 33, where the reagent composition maintains at leastabout 30% activity, as measured by luminescence after the reagentcomposition is combined with the sample, for at least two hours afterthe reagent composition is combined with a sample containing ATP, ascompared to the reagent composition's activity just after the luciferaseis combined with the one or more ATPase inhibitors, and where the one ormore ATPase inhibitors present in the reagent composition arecollectively able to reduce ATPase activity endogenous to the sample byat least about 40% relative to the sample's ATPase activity in theabsence of the one or more ATPase inhibitors.
 38. The kit of claim 33,where the reagent composition maintains at least about 60% activity, asmeasured by luminescence after the reagent composition is combined withthe sample, for at least two hours after the reagent composition iscombined with a sample containing ATP, as compared to the reagentcomposition's activity just after the luciferase is combined with theone or more ATPase inhibitors, and where the one or more ATPaseinhibitors present in the reagent composition are collectively able toreduce ATPase activity endogenous to the sample by at least about 40%relative to the sample's ATPase activity in the absence of the one ormore ATPase inhibitors.
 39. The kit of claim 33, where the reagentcomposition maintains at least about 60% activity, as measured byluminescence after the reagent composition is combined with the sample,for at least one hour after the reagent composition is combined with asample containing ATP, as compared to the reagent composition's activityjust after the luciferase is combined with the one or more ATPaseinhibitors, and where the one or more ATPase inhibitors present in thereagent composition are collectively able to reduce ATPase activityendogenous to the sample by at least about 60% relative to the sample'sATPase activity in the absence of the one or more ATPase inhibitors. 40.The kit of claim 33, where the luciferase in the reagent compositioninitially produces a luminescence that has less than 50% loss ofluminescence during the first hour after the reagent is combined with asample comprising ATP.
 41. The kit of claim 33, where the reagentcomposition comprises at least 0.1% of benzyldimethyldodecylammoniumbromide and maintains at least 50% reagent composition activity duringthe first hour after the reagent is combined with a sample comprisingATP.
 42. The kit of claim 33, where the first container comprises alypholized luciferase in a sealed environment.
 43. The kit of claim 33,where the kit comprises a sterile access port to the first container orthe second container.
 44. A kit for detecting ATP comprising a firstcompartment storing luciferase component comprising a chemostableluciferase separated from a second compartment storing an ATPaseinhibitor component comprising one or more ATPase inhibitors by apartition.
 45. The kit of claim 44, where the partition is a removableor breakable membrane.
 46. The kit of claim 44, where the luciferasecomponent and the ATPase inhibitor component can be admixed by breakingthe partition.
 47. The kit of claim 44, where the partition isremoveable to admix the luciferase component and the ATPase inhibitorcomponent.